Science.gov

Sample records for essential mitogen-activated protein

  1. Heat stress activates the yeast high-osmolarity glycerol mitogen-activated protein kinase pathway, and protein tyrosine phosphatases are essential under heat stress.

    PubMed

    Winkler, Astrid; Arkind, Christopher; Mattison, Christopher P; Burkholder, Anne; Knoche, Kathryn; Ota, Irene

    2002-04-01

    The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1 delta strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways. PMID:12455951

  2. Heat Stress Activates the Yeast High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway, and Protein Tyrosine Phosphatases Are Essential under Heat Stress

    PubMed Central

    Winkler, Astrid; Arkind, Christopher; Mattison, Christopher P.; Burkholder, Anne; Knoche, Kathryn; Ota, Irene

    2002-01-01

    The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1Δ strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways. PMID:12455951

  3. [Mitogen-activated protein kinases in atherosclerosis].

    PubMed

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  4. Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6.

    PubMed Central

    Nakajima, T; Kinoshita, S; Sasagawa, T; Sasaki, K; Naruto, M; Kishimoto, T; Akira, S

    1993-01-01

    NF-IL6, a member of the basic leucine zipper (bZIP) family transcription factors, is involved in expression of inducible genes involved in immune and inflammatory responses. We observed that coexpression of oncogenic p21ras stimulated the transactivating activity of NF-IL6 and induced phosphorylation of Thr-235 located just N-terminal to the DNA binding domain of NF-IL6. Recently, mitogen-activated protein (MAP) kinases have been shown to be implicated in the cellular response to activated ras. Purified MAP kinases specifically phosphorylated Thr-235 of NF-IL6 in vitro. Mutation of Thr-235 abolished the ras-dependent activation of NF-IL6. From these results, we conclude that NF-IL6 is regulated through phosphorylation by MAP kinases in response to activated ras. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8384717

  5. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  6. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  7. Mitogen-activated protein kinases in male reproductive function

    PubMed Central

    Li, Michelle W.M.; Mruk, Dolores D.; Cheng, C. Yan

    2009-01-01

    Recent studies have shown that male reproductive function is modulated via the mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade is involved in numerous male reproductive processes, including spermatogenesis, sperm maturation and activation, capacitation and acrosome reaction, before fertilization of the oocyte. In this review, we discuss the latest findings in this rapidly developing field regarding the role of MAPK in male reproduction in animal models and in human spermatozoa in vitro. This research will facilitate the design of future studies in humans, although much work is needed before this information can be used to manage male infertility and environmental toxicant-induced testicular injury in men, such as blood–testis-barrier disruption. PMID:19303360

  8. Mitogen-activated protein kinase (MAPK) in cardiac tissues.

    PubMed

    Page, C; Doubell, A F

    Mitogen-activated protein kinase (MAPK) has recently emerged as a prominent role player in intracellular signalling in the ventricular myocyte with attention being focussed on its possible role in the development of ventricular hypertrophy. It is becoming clear that MAPK is also active in other cells of cardiac origin such as cardiac fibroblasts and possible functions of this signalling pathway in the heart have yet to be explored. In this report the mammalian MAPK pathway is briefly outlined, before reviewing current knowledge of the MAPK pathway in cardiac tissue (ventricular myocytes, vascular smooth muscle cells and cardiac fibroblasts). New data is also presented on the presence and activity of MAPK in two additional cardiac celltypes namely atrial myocytes and vascular endothelial cells from the coronary microcirculation. PMID:8739228

  9. Protein-protein interactions in plant mitogen-activated protein kinase cascades.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2016-02-01

    Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed. PMID:26646897

  10. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  11. Mitogen activated protein kinase at the nuclear pore complex

    PubMed Central

    Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

    2011-01-01

    Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

  12. Essential oil from the heartwood of Taiwan fir ameliorates LPS-induced inflammatory response by inhibiting the activation of mitogen-activated protein kinase.

    PubMed

    Liu, May-Lan; Hua, Kuo-Feng; Yang, Tzu-Jung; Chiu, Huan-Wen; Ho, Chen-Lung

    2014-10-01

    The essential oil from the heartwood of Taiwan fir (EOTC) was demonstrated to exhibit anti-inflammatory activity in lipopolysaccharide (LPS)-activated mouse macrophages. EOTC reduced nitrite oxide levels and inducible nitrite oxide synthase expression in, and tumor necrosis factor-α and interleukin-6 secretion by, LPS-activated macrophages without affecting cyclooxygenase-2 expression. EOTC reduced the levels of interleukin-lβ precursor induced by LPS and decreased the NLRP3 inflammasome-derived interleukin-lβ secretion induced by LPS and adenosine triphosphate. In addition, the phosphorylation levels of ERKI/2, JNK1/2, and p38 in LPS-activated macrophages were reduced by EOTC. Furthermore, EOTC was composed of oxygenated sesquiterpenes (68.4%), sesquiterpene hydrocarbons (28.9%) and diterpenes (0.9%). The major compounds of the oxygenated sesquiterpenes were τ-cadinol (23.9%), α-cadinol (21.1%) and cedrol (16.9%). These findings suggest that EOTC may be a candidate for the development of anti-inflammatory agents for preventing and ameliorating inflammation-related diseases. PMID:25522551

  13. Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

    PubMed Central

    Corbit, Kevin C.; Foster, David A.; Rosner, Marsha Rich

    1999-01-01

    In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cδ (PKCδ), whereas ERK activation in response to the mitogenic EGF is independent of PKCδ. Antisense PKCδ oligonucleotides or the PKCδ-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCδ functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCδ also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCδ in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCδ requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCδ in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCδ contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling. PMID:10330161

  14. Wounding systemically activates a mitogen-activated protein kinase in forage and turf grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forage and turf grasses are continually cut and grazed by livestock, however very little is known concerning the perception or molecular responses to wounding. Mechanical wounding rapidly activated a 46 kDa and a 44 kDa mitogen-activated protein kinase (MAPK) in six different grass species. In the m...

  15. A novel role for copper in Ras/mitogen-activated protein kinase signaling.

    PubMed

    Turski, Michelle L; Brady, Donita C; Kim, Hyung J; Kim, Byung-Eun; Nose, Yasuhiro; Counter, Christopher M; Winge, Dennis R; Thiele, Dennis J

    2012-04-01

    Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. in vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer. PMID:22290441

  16. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  17. Pivotal Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in Inflammatory Pulmonary Diseases

    PubMed Central

    Qian, Feng; Deng, Jing; Wang, Gang; Ye, Richard D.; Christman, John W.

    2016-01-01

    Mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) is exclusively regulated by p38 MAPK in vivo. Upon activation of p38 MAPK, MK2 binds with p38 MAPK, leading to phosphorylation of TTP, Hsp27, Akt and Cdc25 that are involved in regulation of various essential cellular functions. In this review, we discuss current knowledge about molecular mechanisms of MK2 in regulation of TNF-α production, NADPH oxidase activation, neutrophil migration, and DNA-damage-induced cell cycle arrest which are involved in the molecular pathogenesis of acute lung injury, pulmonary fibrosis, and non-small-cell lung cancer. Collectively current and emerging new information indicate that developing MK2 inhibitors and blocking MK2-mediated signal pathways is a potential therapeutic strategy for treatment of inflammatory and fibrotic lung diseases and lung cancer. PMID:26119506

  18. Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle.

    PubMed

    Trappanese, Danielle M; Sivilich, Sarah; Ets, Hillevi K; Kako, Farah; Autieri, Michael V; Moreland, Robert S

    2016-06-01

    Vascular smooth muscle contraction is primarily regulated by phosphorylation of myosin light chain. There are also modulatory pathways that control the final level of force development. We tested the hypothesis that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase modulate vascular smooth muscle activity via effects on MAP kinase phosphatase-1 (MKP-1). Swine carotid arteries were mounted for isometric force recording and subjected to histamine stimulation in the presence and absence of inhibitors of PKC [bisindolylmaleimide-1 (Bis)], MAP kinase kinase (MEK) (U0126), and MKP-1 (sanguinarine) and flash frozen for measurement of MAP kinase, PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), and caldesmon phosphorylation levels. CPI-17 was phosphorylated in response to histamine and was inhibited in the presence of Bis. Caldesmon phosphorylation levels increased in response to histamine stimulation and were decreased in response to MEK inhibition but were not affected by the addition of Bis. Inhibition of PKC significantly increased p42 MAP kinase, but not p44 MAP kinase. Inhibition of MEK with U0126 inhibited both p42 and p44 MAP kinase activity. Inhibition of MKP-1 with sanguinarine blocked the Bis-dependent increase of MAP kinase activity. Sanguinarine alone increased MAP kinase activity due to its effects on MKP-1. Sanguinarine increased MKP-1 phosphorylation, which was inhibited by inhibition of MAP kinase. This suggests that MAP kinase has a negative feedback role in inhibiting MKP-1 activity. Therefore, PKC catalyzes MKP-1 phosphorylation, which is reversed by MAP kinase. Thus the fine tuning of vascular contraction is due to the concerted effort of PKC, MAP kinase, and MKP-1. PMID:27053523

  19. Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory

    PubMed Central

    Abdul Rahman, Nor Zaihana; Greenwood, Sam M.; Brett, Ros R.; Tossell, Kyoko; Ungless, Mark A.; Plevin, Robin

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2−/− mice), we show that long-term potentiation is impaired in MKP-2−/− mice compared with MKP-2+/+ controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2−/− mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2−/− mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling. SIGNIFICANCE STATEMENT Recently, there has been significant focus on proteins that control mitogen-activated protein kinases' (MAPKs) function, namely the mitogen-activated protein kinase phosphatases (MKPs). Recent studies have revealed novel

  20. Parasite Mitogen-Activated Protein Kinases as Drug Discovery Targets to Treat Human Protozoan Pathogens

    PubMed Central

    Brumlik, Michael J.; Pandeswara, Srilakshmi; Ludwig, Sara M.; Murthy, Kruthi; Curiel, Tyler J.

    2011-01-01

    Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs) as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known. PMID:21637385

  1. Targeting the RAS pathway by mitogen-activated protein kinase inhibitors.

    PubMed

    Kiessling, Michael K; Rogler, Gerhard

    2015-01-01

    Targeting of oncogenic driver mutations with small-molecule inhibitors resulted in powerful treatment options for cancer patients in recent years. The RAS (rat sarcoma) pathway is among the most frequently mutated pathways in human cancer. Whereas targeting mutant Kirsten RAS (KRAS) remains difficult, mutant B rapidly accelerated fibrosarcoma (BRAF) kinase is an established drug target in cancer. Now data show that neuroblastoma RAS (NRAS) and even Harvey RAS (HRAS) mutations could be predictive markers for treatment with mitogen-activated protein kinase (MEK) inhibitors. This review discusses recent preclinical and clinical studies of MEK inhibitors in BRAF and RAS mutant cancer. PMID:26691679

  2. Impacts of Activation of the Mitogen-Activated Protein Kinase Pathway in Pancreatic Cancer

    PubMed Central

    Furukawa, Toru

    2015-01-01

    Pancreatic cancer is characterized by constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. Mutations of KRAS or BRAF and epigenetic abrogation of DUSP6 contribute synergistically to the constitutive activation of MAPK. Active MAPK induces the expression of a variety of genes that are thought to play roles in malignant phenotypes of pancreatic cancer. By blocking the functions of such induced genes, it is possible to attenuate the malignant phenotypes. The development of drugs targeting genes downstream of MAPK may provide a novel therapeutic option for pancreatic cancer. PMID:25699241

  3. Secreted beta-amyloid precursor protein stimulates mitogen-activated protein kinase and enhances tau phosphorylation.

    PubMed Central

    Greenberg, S M; Koo, E H; Selkoe, D J; Qiu, W Q; Kosik, K S

    1994-01-01

    Biological effects related to cell growth, as well as a role in the pathogenesis of Alzheimer disease, have been ascribed to the beta-amyloid precursor protein (beta-APP). Little is known, however, about the intracellular cascades that mediate these effects. We report that the secreted form of beta-APP potently stimulates mitogen-activated protein kinases (MAPKs). Brief exposure of PC-12 pheochromocytoma cells to beta-APP secreted by transfected Chinese hamster ovary cells stimulated the 43-kDa form of MAPK by > 10-fold. Induction of a dominant inhibitory form of ras in a PC12-derived cell line prevented the stimulation of MAPK by secreted beta-APP, demonstrating the dependence of the effect upon p21ras. Because the microtubule-associated protein tau is hyperphosphorylated in Alzheimer disease, we sought and found a 2-fold enhancement in tau phosphorylation associated with the beta-APP-induced MAPK stimulation. In the ras dominant inhibitory cell line, beta-APP failed to enhance phosphorylation of tau. The data presented here provide a link between secreted beta-APP and the phosphorylation state of tau. Images PMID:8041753

  4. 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. PMID:26842916

  5. Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition.

    PubMed

    Lee, Horim

    2015-07-01

    Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition. PMID:26082029

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  8. Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum

    PubMed Central

    Liu, Zhiqin; Shi, Lanping; Liu, Yanyan; Tang, Qian; Shen, Lei; Yang, Sheng; Cai, Jinsen; Yu, Huanxin; Wang, Rongzhang; Wen, Jiayu; Lin, Youquan; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; He, Shuilin

    2015-01-01

    The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper. PMID:26442088

  9. Targeting the mitogen-activated protein kinase pathway in low-grade serous carcinoma of the ovary.

    PubMed

    McLachlan, Jennifer; Gore, Martin; Banerjee, Susana

    2016-08-01

    Until recently, there has been little change in the management of epithelial ovarian cancer with the majority of women receiving identical systemic therapy, regardless of histological subtype. The heterogeneity of epithelial ovarian cancer is now well established, with distinct subtypes characterized by specific molecular alterations and patterns of clinical behavior. Low-grade serous carcinoma is a rare subtype associated with an indolent biological behavior and inherent resistance to chemotherapy. The mitogen-activated protein kinase pathway plays a prominent role in the pathogenesis of low-grade serous carcinoma, and provides an attractive target for novel therapeutic agents. Selumetinib, a MEK1/2 inhibitor, demonstrates promising efficacy in women with relapsed low-grade serous carcinoma, and further trials of MEK-inhibition are underway. Translational research will be essential to identify predictive biomarkers for this treatment approach. PMID:27469379

  10. Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

    PubMed

    Abdul Rahman, Nor Zaihana; Greenwood, Sam M; Brett, Ros R; Tossell, Kyoko; Ungless, Mark A; Plevin, Robin; Bushell, Trevor J

    2016-02-24

    Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling. PMID:26911683

  11. Mitogen-Activated Protein Kinase Phosphatase 2 Regulates the Inflammatory Response in Sepsis▿

    PubMed Central

    Cornell, Timothy T.; Rodenhouse, Paul; Cai, Qing; Sun, Lei; Shanley, Thomas P.

    2010-01-01

    Sepsis results from a dysregulation of the regulatory mechanisms of the pro- and anti-inflammatory response to invading pathogens. The mitogen-activated protein (MAP) kinase cascades are key signal transduction pathways involved in the cellular production of cytokines. The dual-specific phosphatase 1 (DUSP 1), mitogen-activated protein kinase phosphatase-1 (MKP-1), has been shown to be an important negative regulator of the inflammatory response by regulating the p38 and Jun N-terminal protein kinase (JNK) MAP kinase pathways to influence pro- and anti-inflammatory cytokine production. MKP-2, also a dual-specific phosphatase (DUSP 4), is a phosphatase highly homologous with MKP-1 and is known to regulate MAP kinase signaling; however, its role in regulating the inflammatory response is not known. We hypothesized a regulatory role for MKP-2 in the setting of sepsis. Mice lacking the MKP-2 gene had a survival advantage over wild-type mice when challenged with intraperitoneal lipopolysaccharide (LPS) or a polymicrobial infection via cecal ligation and puncture. The MKP-2−/− mice also exhibited decreased serum levels of both pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], IL-6) and anti-inflammatory cytokines (IL-10) following endotoxin challenge. Isolated bone marrow-derived macrophages (BMDMs) from MKP-2−/− mice showed increased phosphorylation of the extracellular signal-regulated kinase (ERK), decreased phosphorylation of JNK and p38, and increased induction of MKP-1 following LPS stimulation. The capacity for cytokine production increased in MKP-2−/− BMDMs following MKP-1 knockdown. These data support a mechanism by which MKP-2 targets ERK deactivation, thereby decreasing MKP-1 and thus removing the negative inhibition of MKP-1 on cytokine production. PMID:20351138

  12. Crosstalk and Signaling Switches in Mitogen-Activated Protein Kinase Cascades

    PubMed Central

    Fey, Dirk; Croucher, David R.; Kolch, Walter; Kholodenko, Boris N.

    2012-01-01

    Mitogen-activated protein kinase (MAPK) cascades control cell fate decisions, such as proliferation, differentiation, and apoptosis by integrating and processing intra- and extracellular cues. However, similar MAPK kinetic profiles can be associated with opposing cellular decisions depending on cell type, signal strength, and dynamics. This implies that signaling by each individual MAPK cascade has to be considered in the context of the entire MAPK network. Here, we develop a dynamic model of feedback and crosstalk for the three major MAPK cascades; extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), c-Jun N-terminal kinase (JNK), and also include input from protein kinase B (AKT) signaling. Focusing on the bistable activation characteristics of the JNK pathway, this model explains how pathway crosstalk harmonizes different MAPK responses resulting in pivotal cell fate decisions. We show that JNK can switch from a transient to sustained activity due to multiple positive feedback loops. Once activated, positive feedback locks JNK in a highly active state and promotes cell death. The switch is modulated by the ERK, p38, and AKT pathways. ERK activation enhances the dual specificity phosphatase (DUSP) mediated dephosphorylation of JNK and shifts the threshold of the apoptotic switch to higher inputs. Activation of p38 restores the threshold by inhibiting ERK activity via the PP1 or PP2A phosphatases. Finally, AKT activation inhibits the JNK positive feedback, thus abrogating the apoptotic switch and allowing only proliferative signaling. Our model facilitates understanding of how cancerous deregulations disturb MAPK signal processing and provides explanations for certain drug resistances. We highlight a critical role of DUSP1 and DUSP2 expression patterns in facilitating the switching of JNK activity and show how oncogene induced ERK hyperactivity prevents the normal apoptotic switch explaining the failure of certain drugs to

  13. The case for inhibiting p38 mitogen-activated protein kinase in heart failure

    PubMed Central

    Arabacilar, Pelin; Marber, Michael

    2015-01-01

    This minireview discusses the evidence that the inhibition of p38 mitogen-activated protein kinases (p38 MAPKs) maybe of therapeutic value in heart failure. Most previous experimental studies, as well as past and ongoing clinical trials, have focussed on the role of p38 MAPKs in myocardial infarction and acute coronary syndromes. There is now growing evidence that these kinases are activated within the myocardium of the failing human heart and in the heart and blood vessels of animal models of heart failure. Furthermore, from a philosophical viewpoint the chronic activation of the adaptive stress pathways that lead to the activation of p38 MAPKs in heart failure is analogous to the chronic activation of the sympathetic, renin-aldosterone-angiotensin and neprilysin systems. These have provided some of the most effective therapies for heart failure. This minireview questions whether similar and synergistic advantages would follow the inhibition of p38 MAPKs. PMID:26029107

  14. Mitogen-Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical Interplay in Macrophage Biology

    PubMed Central

    Lloberas, Jorge; Valverde-Estrella, Lorena; Tur, Juan; Vico, Tania; Celada, Antonio

    2016-01-01

    Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation. PMID:27446931

  15. p38 mitogen-activated protein kinase inhibitor reduces neurocan production in cultured spinal cord astrocytes.

    PubMed

    Yamaoka, Gotaro; Morino, Tadao; Morizane, Kei; Horiuchi, Hideki; Miura, Hiromasa; Ogata, Tadanori

    2012-06-20

    Chondroitin sulfate proteoglycans are formed in scar tissue after a spinal cord injury and inhibit axon regrowth. The production of neurocan, one of these chondroitin sulfate proteoglycans, in cultured spinal cord astrocytes increased after the addition of epidermal growth factor (EGF) in a dose-dependent manner (2-200 ng/ml). In astrocytes stimulated by 20 ng/ml of EGF, neurocan production was inhibited after the addition of the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580: 3-10 μM) in a dose-dependent manner. These results suggest that the activation of p38 MAPK is one of the mechanisms of neurocan production in EGF-stimulated astrocytes. The p38 MAPK inhibitor may reduce neurocan production and accelerate axonal regrowth after a spinal cord injury. PMID:22525836

  16. Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells.

    PubMed

    Khalil, R A; Menice, C B; Wang, C L; Morgan, K G

    1995-06-01

    Tyrosine phosphorylation has been linked to plasmalemmal targeting of src homology-2-containing proteins, activation of mitogen-activated protein (MAP) kinase, nuclear signaling, and proliferation of cultured cells. Significant tyrosine phosphorylation and MAP kinase activities have also been reported in differentiated cells, but the signaling role of tyrosine-phosphorylated MAP kinase in these cells is unclear. The spatial and temporal relation between phosphotyrosine and MAP kinase immunoreactivity was quantified in differentiated contractile vascular smooth muscle cells by using digital imaging microscopy. An initial association of MAP kinase with the plasmalemma required upstream protein kinase C activity but occurred in a tyrosine phosphorylation-independent manner. Subsequent to membrane association, a delayed redistribution of MAP kinase, colocalizing with the actin-binding protein caldesmon, occurred in a tyrosine phosphorylation-dependent manner. The apparent association of MAP kinase with the contractile proteins coincided with contractile activation. Thus, tyrosine phosphorylation appears to target MAP kinase to cytoskeletal proteins in contractile vascular cells. This targeting mechanism may determine the specific destination and thereby the specialized function of MAP kinase in other phenotypes. PMID:7538916

  17. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana.

    PubMed

    Sheikh, Arsheed H; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  18. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana

    PubMed Central

    Sheikh, Arsheed H.; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K.; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  19. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana

    PubMed Central

    Lassowskat, Ines; Böttcher, Christoph; Eschen-Lippold, Lennart; Scheel, Dierk; Lee, Justin

    2014-01-01

    Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the “PEN” pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org). PMID:25368622

  20. 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

  1. Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases.

    PubMed

    Dayalan Naidu, Sharadha; Sutherland, Calum; Zhang, Ying; Risco, Ana; de la Vega, Laureano; Caunt, Christopher J; Hastie, C James; Lamont, Douglas J; Torrente, Laura; Chowdhry, Sudhir; Benjamin, Ivor J; Keyse, Stephen M; Cuenda, Ana; Dinkova-Kostova, Albena T

    2016-09-15

    Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response. PMID:27354066

  2. Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases

    PubMed Central

    Dayalan Naidu, Sharadha; Sutherland, Calum; Zhang, Ying; Risco, Ana; de la Vega, Laureano; Caunt, Christopher J.; Hastie, C. James; Lamont, Douglas J.; Torrente, Laura; Chowdhry, Sudhir; Benjamin, Ivor J.; Keyse, Stephen M.; Cuenda, Ana

    2016-01-01

    Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response. PMID:27354066

  3. Mitogen-activated protein kinase phosphatase-1: function and regulation in bone and related tissues.

    PubMed

    Broome, David T; Datta, Nabanita S

    2016-05-01

    In this review, we have highlighted work that has clearly demonstrated that mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), a negative regulator of MAPKs, is an important signaling mediator in bone, muscle, and fat tissue homeostasis and differentiation. Further, we examined recent studies with particular focus on MKP-1 overexpression or deletion and its impact on tissues connected to bone. We also summarized regulation of MKP-1 by known skeletal regulators like parathyroid hormone (PTH)/PTH-related peptide (PTHrP) and bone morphogenic proteins. MKP-1's integration into the pathophysiological state of osteoporosis, osteoarthritis, rheumatoid arthritis, obesity, and muscular dystrophy are examined to emphasize possible involvement of MKP-1 both at the molecular level and in disease complications such as sarcopenia- or diabetes-related osteoporosis. We predict that understanding the mechanism of MKP-1-mediated signaling in bone-muscle-fat crosstalk will be a key in coordinating their activities and developing therapeutics to improve clinical outcomes for diseases associated with advanced age. PMID:27031422

  4. Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase.

    PubMed

    Mendoza-Mendoza, Artemio; Pozo, María J; Grzegorski, Darlene; Martínez, Pedro; García, Juan M; Olmedo-Monfil, Vianey; Cortés, Carlos; Kenerley, Charles; Herrera-Estrella, Alfredo

    2003-12-23

    The production of lytic enzymes in Trichoderma is considered determinant in its parasitic response against fungal species. A mitogen-activated protein kinase encoding gene, tvk1, from Trichoderma virens was cloned, and its role during the mycoparasitism, conidiation, and biocontrol was examined in tvk1 null mutants. These mutants showed a clear increase in the level of the expression of mycoparasitism-related genes under simulated mycoparasitism and during direct confrontation with the plant pathogen Rhizoctonia solani. The null mutants displayed an increased protein secretion phenotype as measured by the production of lytic enzymes in culture supernatant compared to the wild type. Consistently, biocontrol assays demonstrated that the null mutants were considerably more effective in disease control than the wild-type strain or a chemical fungicide. In addition, tvk1 gene disruptant strains sporulated abundantly in submerged cultures, a condition that is not conducive to sporulation in the wild type. These data suggest that Tvk1 acts as a negative modulator during host sensing and sporulation in T. virens. PMID:14673101

  5. Dermatophytes Activate Skin Keratinocytes via Mitogen-Activated Protein Kinase Signaling and Induce Immune Responses

    PubMed Central

    Achterman, Rebecca R.; Moyes, David L.; Thavaraj, Selvam; Smith, Adam R.; Blair, Kris M.

    2015-01-01

    Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation. PMID:25667269

  6. A Novel Mitogen-Activated Protein Kinase Is Responsive to Raf and Mediates Growth Factor Specificity

    PubMed Central

    Janulis, Mark; Trakul, Nicholas; Greene, Geoffrey; Schaefer, Erik M.; Lee, J. D.; Rosner, Marsha Rich

    2001-01-01

    The proto-oncogene Raf is a major regulator of growth and differentiation. Previous studies from a number of laboratories indicate that Raf activates a signaling pathway that is independent of the classic MEK1,2-ERK1,2 cascade. However, no other signaling cascade downstream of Raf has been identified. We describe a new member of the mitogen-activated protein kinase family, p97, an ERK5-related kinase that is activated and Raf associated when cells are stimulated by Raf. Furthermore, p97 is selectively responsive to different growth factors, providing a mechanism for specificity in cellular signaling. Thus, p97 is activated by the neurogenic factor fibroblast growth factor (FGF) but not the mitogenic factor epidermal growth factor (EGF) in neuronal cells. Conversely, the related kinase ERK5 is activated by EGF but not FGF. p97 phosphorylates transcription factors such as Elk-1 and Ets-2 but not MEF2C at transactivating sites, whereas ERK5 phosphorylates MEF2C but not Elk-1 or Ets-2. Finally, p97 is expressed in a number of cell types including primary neural and NIH 3T3 cells. Taken together, these results identify a new signaling pathway that is distinct from the classic Raf-MEK1,2-ERK1,2 kinase cascade and can be selectively stimulated by growth factors that produce discrete biological outcomes. PMID:11238956

  7. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    NASA Astrophysics Data System (ADS)

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-06-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets.

  8. Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae

    PubMed Central

    Raina, Susheel Kumar; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna

    2013-01-01

    Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tolerance to abiotic stresses. Yeast cells transformed with CrMPK3 was found to show enhanced tolerance to UV and heat stress. Comparison of CrMPK3 and SLT2, a MAPK from yeast shows high-sequence identity particularly at conserved domains. Additionally, heat stress is also shown to activate a 43 kDa MAP kinase, possibly CrMPK3 in C. roseus leaves. These findings indicate the role of CrMPK3 in stress-induced MIA accumulation as well as in stress tolerance. PMID:23221751

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

    PubMed Central

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

    2015-01-01

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

  10. 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

  11. Flavonoids inhibit iNOS production via mitogen activated proteins in lipoteichoic acid stimulated cardiomyoblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Ventura-Arroyo, Jairo Agustín; Arreguín-Cano, Juan Antonio; Ostoa-Pérez, María Fernanda

    2014-08-01

    Infective endocarditis is caused by oral commensal bacteria which are important etiologic agents in this disease and can induce release of nitric oxide (NO), promoting an inflammatory response in the endocardium. In this study, we investigated the properties of kaempherol, epigallocatechin, apigenin, and naringin in embryonic mouse heart cells (H9c2) treated with lipoteichoic acid (LTA) obtained from Streptococcus sanguinis. NO production was measured with the Griess method. Expression of inducible nitric oxide synthase (iNOS) was detected by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, western blot assays and immunofluorescence staining were used to assess translocation of nuclear factor kappa beta (NF-κB), degradation of IκB, and activity of the mitogen activated protein (MAP) kinases extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun N-terminal kinase (JNK). And the effects of these flavonoids on cell viability were also assessed. Our results showed that flavonoids blocked activation of ERK, JNK, and p38 in cardiomyocytes treated with LTA. Moreover, the flavonoids showed no cytotoxic effects and blocked NF-κB translocation and IκB degradation and inhibited LTA-induced NF-κB promoter activity, iNOS expression and NO production. In conclusion these effects are consistent with some of the observed anti-inflammatory properties of other flavonoids. PMID:24768712

  12. Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species.

    PubMed

    Haase, Hajo; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar

    2011-01-01

    Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg(2+) ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn(2+)); and (3) by inducing reactive oxygen species (ROS). Hg(2+) causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn(2+) release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn(2+) or Hg(2+). Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg(2+)-induced oxidation, because phosphatase activity is inhibited at concentrations of Hg(2+) that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system. PMID:20951154

  13. Crosstalk between mitogen-activated protein kinases and mitochondria in cardiac diseases: therapeutic perspectives

    PubMed Central

    Javadov, Sabzali; Jang, Sehwan; Agostini, Bryan

    2014-01-01

    Cardiovascular diseases cause more mortality and morbidity worldwide than any other diseases. Although many intracellular signaling pathways influence cardiac physiology and pathology, the mitogen-activated protein kinase (MAPK) family has garnered significant attention because of its vast implications in signaling and cross-talk with other signaling networks. The extensively studied MAPKs ERK1/2, p38, JNK, and ERK5, demonstrate unique intracellular signaling mechanisms, responding to a myriad of mitogens and stressors and influencing the signaling of cardiac development, metabolism, performance, and pathogenesis. Definitive relationships between MAPK signaling and cardiac dysfunction remain elusive, despite 30 years of extensive clinical studies and basic research of various animal/cell models, severities of stress, and types of stimuli. Still, several studies have proven the importance of MAPK cross-talk with mitochondria, powerhouses of the cell that provide over 80% of ATP for normal cardiomyocyte function and play a crucial role in cell death. Although many questions remain unanswered, there exists enough evidence to consider the possibility of targeting MAPK-mitochondria interactions in the prevention and treatment of heart disease. The goal of this review is to integrate previous studies into a discussion of MAPKs and MAPK-mitochondria signaling in cardiac diseases, such as myocardial infarction (ischemia), hypertrophy and heart failure. A comprehensive understanding of relevant molecular mechanisms, as well as challenges for studies in this area, will facilitate the development of new pharmacological agents and genetic manipulations for therapy of cardiovascular diseases. PMID:24924700

  14. Role of Mitogen-Activated Protein Kinases in Myocardial Ischemia-Reperfusion Injury during Heart Transplantation

    PubMed Central

    Vassalli, Giuseppe; Milano, Giuseppina; Moccetti, Tiziano

    2012-01-01

    In solid organ transplantation, ischemia/reperfusion (IR) injury during organ procurement, storage and reperfusion is an unavoidable detrimental event for the graft, as it amplifies graft inflammation and rejection. Intracellular mitogen-activated protein kinase (MAPK) signaling pathways regulate inflammation and cell survival during IR injury. The four best-characterized MAPK subfamilies are the c-Jun NH2-terminal kinase (JNK), extracellular signal- regulated kinase-1/2 (ERK1/2), p38 MAPK, and big MAPK-1 (BMK1/ERK5). Here, we review the role of MAPK activation during myocardial IR injury as it occurs during heart transplantation. Most of our current knowledge regarding MAPK activation and cardioprotection comes from studies of preconditioning and postconditioning in nontransplanted hearts. JNK and p38 MAPK activation contributes to myocardial IR injury after prolonged hypothermic storage. p38 MAPK inhibition improves cardiac function after cold storage, rewarming and reperfusion. Small-molecule p38 MAPK inhibitors have been tested clinically in patients with chronic inflammatory diseases, but not in transplanted patients, so far. Organ transplantation offers the opportunity of starting a preconditioning treatment before organ procurement or during cold storage, thus modulating early events in IR injury. Future studies will need to evaluate combined strategies including p38 MAPK and/or JNK inhibition, ERK1/2 activation, pre- or postconditioning protocols, new storage solutions, and gentle reperfusion. PMID:22530110

  15. p38 Mitogen-Activated Protein Kinase Pathway Regulates Genes during Proliferation and Differentiation in Oligodendrocytes

    PubMed Central

    Haines, Jeffery D.; Fulton, Debra L.; Richard, Stephane; Almazan, Guillermina

    2015-01-01

    We have previously shown that p38 mitogen-activated protein kinase (p38 MAPK) is important for oligodendrocyte (OLG) differentiation and myelination. However, the precise cellular mechanisms by which p38 regulates OLG differentiation remain largely unknown. To determine whether p38 functions in part through transcriptional events in regulating OLG identity, we performed microarray analysis on differentiating oligodendrocyte progenitors (OLPs) treated with a p38 inhibitor. Consistent with a role in OLG differentiation, pharmacological inhibition of p38 down-regulated the transcription of genes that are involved in myelin biogenesis, transcriptional control and cell cycle. Proliferation assays showed that OLPs treated with the p38 inhibitor retained a proliferative capacity which could be induced upon application of mitogens demonstrating that after two days of p38-inhibition OLGs remained poised to continue mitosis. Together, our results suggest that the p38 pathway regulates gene transcription which can coordinate OLG differentiation. Our microarray dataset will provide a useful resource for future studies investigating the molecular mechanisms by which p38 regulates oligodendrocyte differentiation and myelination. PMID:26714323

  16. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    PubMed Central

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-01-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets. PMID:23822503

  17. Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in Sertoli cells

    PubMed Central

    Fix, Charity; Jordan, Cynthia; Cano, Patricia; Walker, William H.

    2004-01-01

    The androgen testosterone is essential for the Sertoli cell to support the maturation of male germ cells and the production of spermatozoa (spermatogenesis). In the classical view of androgen action, binding of androgen to the intracellular androgen receptor (AR) produces a conformational change in AR such that the receptor–steroid complex has high affinity for specific DNA regulatory elements and is able to stimulate gene transcription. Here, we demonstrate that testosterone can act by means of an alternative, rapid, and sustainable mechanism in Sertoli cells that is independent of AR–DNA interactions. Specifically, the addition of physiological levels of testosterone to Sertoli cells stimulates the mitogen-activated protein kinase signaling pathway and causes phosphorylation of the cAMP response element binding protein transcription factor on serine 133, a modification known to be required for Sertoli cells to support spermatogenesis. Androgen-mediated activation of mitogen-activated protein kinase and cAMP response element binding protein occurs within 1 min, extends for at least 12 h and requires AR. Furthermore, androgen induces endogenous cAMP response element binding protein-mediated transcription in Sertoli cells. These newly identified mechanisms of androgen action in Sertoli cells suggest new targets for developing male contraceptive agents. PMID:15263086

  18. Azorella compacta methanolic extract induces apoptosis via activation of mitogen-activated protein kinase.

    PubMed

    Sung, Min Hee; Kwon, Ok-Kyoung; Oh, Sei-Ryang; Lee, Joongku; Park, Sang-Hong; Han, Sang Bae; Ahn, Kyung-Seop

    2015-11-01

    Azorella compacta Phil. (AC) is an alpine medicinal plant used traditionally for antibacterial treatment. Recent studies have revealed that this plant also has anti‑diabetic effects, but that it is toxic. The present study investigated the underlying mechanisms of action of AC extract against human leukemia HL60 cells. Apoptosis induction was measured by MTT assay, fluorescence microscopy, DNA fragmentation assay, flow cytometric analysis, reverse transcription quantitative polymerase chain reaction and western blot analyses. It was found that AC extract inhibited the growth of HL60 and other cancer cell lines in a dose‑dependent manner. The cytotoxic effects of AC extract on HL60 cells were associated with apoptosis characterized by DNA fragmentation and dose‑dependent increases in Annexin V‑positive cells, as determined by flow cytometric analysis. AC‑extract‑induced apoptosis was accompanied by activated/cleaved caspase‑3, caspase‑9 and poly(adenosine diphosphate‑ribose) polymerase (PARP). The increases in apoptosis were also associated with decreases of the apoptosis-inhibitor B-cell lymphoma 2 (Bcl‑2), upregulation of pro‑apoptotic Bcl-2-associated X (Bax) protein and downregulation of anti‑apoptotic Bcl extra large protein. Furthermore, western blot analysis of mitogen-activated protein kinase (MAPK)-associated proteins indicated that treatment with AC extract increased the levels of c-Jun N-terminal kinase, extracellular signal-regulated kinase and p38. In addition, the expression of Bax and cleaved PARP was blocked when AC treatment was performed in the presence of MAPK inhibitors. It was therefore concluded that AC induced apoptosis in human leukemia HL60 cells via an intrinsic pathway controlled through MAPK-associated signaling. PMID:26397193

  19. Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages.

    PubMed Central

    Büscher, D; Hipskind, R A; Krautwald, S; Reimann, T; Baccarini, M

    1995-01-01

    Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation. PMID:7799956

  20. Molecular pathways: mitogen-activated protein kinase pathway mutations and drug resistance.

    PubMed

    Pritchard, Antonia L; Hayward, Nicholas K

    2013-05-01

    Receptor tyrosine kinases are a diverse family of transmembrane proteins that can activate multiple pathways upon ligation of the receptor, one of which is the series of mitogen-activated protein kinase (MAPK) signaling cascades. The MAPK pathways play critical roles in a wide variety of cancer types, from hematologic malignancies to solid tumors. Aberrations include altered expression levels and activation states of pathway components, which can sometimes be attributable to mutations in individual members. The V600E mutation of BRAF was initially described in 2002 and has been found at particularly high frequency in melanoma and certain subtypes of colorectal cancer. In the relatively short time since this discovery, a family of drugs has been developed that specifically target this mutated BRAF isoform, which, after results from phase I/II and III clinical trials, was granted U.S. Food and Drug Administration approval in August 2011. Although these drugs produce clinically meaningful increases in progression-free and overall survival, due to acquired resistance they have not improved mortality rates. New drugs targeting other members of the MAPK pathways are in clinical trials or advanced stages of development. It is hoped that combination therapies of these new drugs in conjunction with BRAF inhibitors will counteract the mechanisms of resistance and provide cures. The clinical implementation of next-generation sequencing is leading to a greater understanding of the genetic architecture of tumors, along with acquired mechanisms of drug resistance, which will guide the development of tumor-specific inhibitors and combination therapies in the future. PMID:23406774

  1. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells

    PubMed Central

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-01-01

    Background Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. Material/Methods We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) – MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) – were detected by Western blot. Results Results showed that TSLP promoted the production of α-SMA and collagen I (P<0.001), suggesting that it can accelerate MRC-5 cell fibrosis. It also activated the expression of MAPK7, p-p38, p-ERK1, and p-JNK1, but the total MAPK7, p-38, ERK1, and JNK1 protein levels were mostly unchanged, indicating the activated MAPK pathways that might contribute to the promotion of cell fibrosis. Conclusions This study shows the pro-fibrotic role of TSLP in MRC-5 cells, suggesting TSLP is a potential therapeutic target for treating lung fibrosis in ALI. It possibly functions via activating MAPKs. These findings add to our understanding of the mechanism of fibrosis. PMID:27385084

  2. A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

    PubMed Central

    Wang, Xiao Min; Zhai, Ye; Ferrell, James E.

    1997-01-01

    The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells. PMID:9128253

  3. Inactivation of mitogen-activated protein kinase signaling pathway reduces caspase-14 expression in impaired keratinocytes

    PubMed Central

    Dang, Ningning; Pang, Shuguang; Song, Haiyan; An, Liguo; Ma, Xiaoli

    2016-01-01

    Objective(s): Several investigations have revealed that caspase-14 is responsible for the epidermal differentiation and cornification, as well as the regulation of moisturizing effect. However, the precise regulation mechanism is still not clear. This study was aimed to investigate the expression of caspase-14 in filaggrin-deficient normal human epidermal keratinocytes (NHEKs) and to explore the possible mechanism that contributes to the regulation of caspase-14. Materials and Methods: The filaggrin-deficient NHEKs were induced by transfection with lentivirus (LV) vector encoding small hairpin RNAs (shRNA). The inhibitors SB203580, PD98059 and SP600125 were used for suppressing the expression of p38 mitogen-activated protein kinase (MAPK), p44/42 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). The expression of filaggrin, p38 MAPK, p44/42 MAPK and SAPK/JNK, caspase-14, keratin1and keratin2 were detected by western blot. Results: In filaggrin-deficient NHEKs, the expression of p38, p44/42 MAPK and SAPK/JNK and caspase-14 were significantly decreased. The inhibition of p38 and SAPK/JNK reduced the expression of caspase-14, while the p44/42 MAPK showed no consistent effects. Moreover, the filaggrin knockdown decreased the expression of keratin2, but had no effects on the level of keratin1. Conclusion: The decreased expression of caspase-14 in filaggrin-deficient NHEKs may be induced by the inactivation of MAPK signaling pathway. These provide a novel perspective to understand the mechanism for the protective effects of filaggrin and caspase-14 on skin barrier function. PMID:27096061

  4. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells.

    PubMed

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-01-01

    BACKGROUND Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. MATERIAL AND METHODS We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) - MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) - were detected by Western blot. RESULTS Results showed that TSLP promoted the production of α-SMA and collagen I (P<0.001), suggesting that it can accelerate MRC-5 cell fibrosis. It also activated the expression of MAPK7, p-p38, p-ERK1, and p-JNK1, but the total MAPK7, p-38, ERK1, and JNK1 protein levels were mostly unchanged, indicating the activated MAPK pathways that might contribute to the promotion of cell fibrosis. CONCLUSIONS This study shows the pro-fibrotic role of TSLP in MRC-5 cells, suggesting TSLP is a potential therapeutic target for treating lung fibrosis in ALI. It possibly functions via activating MAPKs. These findings add to our understanding of the mechanism of fibrosis. PMID:27385084

  5. Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

    PubMed Central

    Mandal, Goutam; Sharma, Mansi; Kruse, Martin; Sander-Juelch, Claudia; Munro, Laura Anne; Wang, Yong; Vilg, Jenny Veide; Tamás, Markus J; Bhattacharjee, Hiranmoy; Wiese, Martin; Mukhopadhyay, Rita

    2012-01-01

    Summary Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defense against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites co-expressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr197 and this phosphorylation requires LmjMPK2 activity. Lys42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. L. mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a MAP kinase. PMID:22779703

  6. Inhibition of the mitogen-activated protein kinase pathway triggers B16 melanoma cell differentiation.

    PubMed

    Englaro, W; Bertolotto, C; Buscà, R; Brunet, A; Pagès, G; Ortonne, J P; Ballotti, R

    1998-04-17

    In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clément, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21(ras) and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21(ras) and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents. PMID:9545341

  7. Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

    SciTech Connect

    Haase, Hajo; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar

    2011-01-01

    Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg{sup 2+} ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn{sup 2+}); and (3) by inducing reactive oxygen species (ROS). Hg{sup 2+} causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn{sup 2+} release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn{sup 2+} or Hg{sup 2+}. Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg{sup 2+}-induced oxidation, because phosphatase activity is inhibited at concentrations of Hg{sup 2+} that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

  8. Activation of p38 Mitogen-Activated Protein Kinase Promotes Peritoneal Fibrosis by Regulating Fibrocytes

    PubMed Central

    Kokubo, Satoshi; Sakai, Norihiko; Furuichi, Kengo; Toyama, Tadashi; Kitajima, Shinji; Okumura, Toshiya; Matsushima, Kouji; Kaneko, Shuichi; Wada, Takashi

    2012-01-01

    ♦ Background: Peritoneal fibrosis is a serious complication of long-term peritoneal dialysis, and yet the precise pathogenic mechanisms of peritoneal fibrosis remain unknown. Fibrocytes participate in tissue fibrosis and express chemokine receptors that are necessary for migration. The p38 mitogen-activated protein kinase (MAPK) pathway regulates the production of chemokines and has been demonstrated to contribute to the pathogenesis of various fibrotic conditions. Accordingly, we used an experimental mouse model of peritoneal fibrosis to examine the dependency of fibrocytes on p38MAPK signaling. ♦ Methods: Peritoneal fibrosis was induced in mice by the injection of 0.1% chlorhexidine gluconate (CG) into the abdominal cavity. Mice were treated with FR167653, a specific inhibitor of p38MAPK, and immunohistochemical studies were performed to detect fibrocytes and cells positive for phosphorylated p38MAPK. The involvement of p38MAPK in the activation of fibrocytes also was also investigated in vitro. ♦ Results: Fibrocytes infiltrated peritoneum in response to CG, and that response was accompanied by progressive peritoneal fibrosis. The phosphorylation of p38MAPK, as defined by CD45+ spindle-shaped cells, was detected both in peritoneal mesothelial cells and in fibrocytes. The level of peritoneal expression of CCL2, a chemoattractant for fibrocytes, was upregulated by CG injection, and treatment with FR167653 reduced the number of cells positive for phosphorylated p38MAPK, the peritoneal expression of CCL2, and the extent of peritoneal fibrosis. Pretreatment with FR167653 inhibited the expression of procollagen type I α1 induced by transforming growth factor-β1. ♦ Conclusions: Our results suggest that p38MAPK signaling contributes to peritoneal fibrosis by regulating fibrocyte function. PMID:21719683

  9. Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

    PubMed

    Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

    2016-03-21

    Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. PMID:26948880

  10. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

    PubMed Central

    Alagramam, Kumar N.; Stepanyan, Ruben; Jamesdaniel, Samson; Chen, Daniel H.-C.; Davis, Rickie R.

    2015-01-01

    Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4–8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b, Cacna1g, and Pla2g6, related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway. PMID:25387536

  11. Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

    PubMed Central

    Peraldi, P; Zhao, Z; Filloux, C; Fischer, E H; Van Obberghen, E

    1994-01-01

    Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP. Images PMID:8197172

  12. JNK and p38 mitogen-activated protein kinase pathways contribute to porcine epidemic diarrhea virus infection.

    PubMed

    Lee, Changhee; Kim, Youngnam; Jeon, Ji Hyun

    2016-08-15

    The mitogen-activated protein kinase (MAPK) pathways, which are central building blocks in the intracellular signaling network, are often manipulated by viruses of diverse families to favor their replication. Among the MAPK family, the extracellular signal-regulated kinase (ERK) pathway is known to be modulated during the infection with porcine epidemic diarrhea virus (PEDV); however, involvement of stress-activated protein kinases (SAPKs) comprising p38 MAPK and c-Jun NH2-terminal kinase (JNK) remains to be determined. Therefore, in the present study, we investigated whether activation of p38 MAPK and JNK cascades is required for PEDV replication. Our results showed that PEDV activates p38 MAPK and JNK1/2 up to 24h post-infection, whereas, thereafter their phosphorylation levels recede to baseline levels or even fall below them. Notably, UV-irradiated inactivated PEDV, which can enter cells but cannot replicate inside them, failed to induce phosphorylation of p38 MAPK and JNK1/2 suggesting that viral biosynthesis is essential for activation of these kinases. Treatment of cells with selective p38 or JNK inhibitors markedly impaired PEDV replication in a dose-dependent manner and these antiviral effects were found to be maximal during the early times of the infection. Furthermore, direct pharmacological inhibition of p38 MAPK or JNK1/2 activation resulted in a significant reduction of viral RNA synthesis, viral protein expression, and progeny release. However, independent treatments with either SAPK inhibitor did not inhibit PEDV-induced apoptotic cell death mediated by activation of mitochondrial apoptosis-inducing factor (AIF) suggesting that SAPKs are irrelevant to the apoptosis pathway during PEDV infection. In summary, our data demonstrated critical roles of the p38 and JNK1/2 signaling pathways in facilitating successful viral infection during the post-entry steps of the PEDV life cycle. PMID:27215486

  13. Mitogen Activated Protein Kinase Family Proteins and c-jun Signaling in Injury-induced Schwann Cell Plasticity.

    PubMed

    Lee, Hye Jeong; Shin, Yoon Kyung; Park, Hwan Tae

    2014-06-01

    Schwann cells (SCs) in the peripheral nerves myelinate axons during postnatal development to allow saltatory conduction of nerve impulses. Well-organized structures of myelin sheathes are maintained throughout life unless nerves are insulted. After peripheral nerve injury, unidentified signals from injured nerves drive SC dedifferentiation into an immature state. Dedifferentiated SCs participate in axonal regeneration by producing neurotrophic factors and removing degenerating nerve debris. In this review, we focus on the role of mitogen activated protein kinase family proteins (MAP kinases) in SC dedifferentiation. In addition, we will highlight neuregulin 1 and the transcription factor c-jun as upstream and downstream signals for MAP kinases in SC responses to nerve injury. PMID:24963277

  14. Involvement of the mitogen activated protein kinase Hog1p in the response of Candida albicans to iron availability

    PubMed Central

    2013-01-01

    Background Iron is an essential nutrient for almost all organisms, and generating iron limiting conditions for pathogens is one of the host defense strategies against microbial infections. Excess of iron can be toxic; therefore, iron uptake is tightly controlled. The high affinity iron uptake system of the opportunistic pathogenic yeast Candida albicans has been shown to be essential for virulence. Several transcription factors and regulators of iron uptake genes were identified, but the knowledge of signaling pathways is still limited. Gene expression profiling of the Δhog1 deletion mutant indicated an involvement of the mitogen activated protein (MAP) kinase Hog1p. However, the function of Hog1p in the response of C. albicans to iron availability was not studied in detail. Thus, we analyzed phenotypic and molecular responses of C. albicans to different iron concentrations particularly with respect to the activity of the Hog1p MAP kinase module. Results We observed flocculation of yeast cells, when the iron ion concentration was equal to or higher than 5 μM. This phenotype was dependent on the MAP kinase Hog1p and the corresponding MAP kinase kinase Pbs2p. Moreover, high extracellular iron ion concentrations led to hyper-phosphorylation of Hog1p. We determined lower amounts of multicopper ferroxidase (MCFO) proteins and lower ferric reductase activity, when the iron ion concentration in the medium was increased. This effect was also observed for the Δhog1 mutant. However, the amounts of MCFO proteins and the cell surface ferric reductase activity were increased in the Δhog1 in comparison to wild type cells. This effect was independent of iron availability in growth media. Conclusions In C. albicans, the MAP kinase Hog1p is part of the network regulating the response of the organism to iron availability. Hog1p was transiently phosphorylated under high iron concentrations and was essential for a flocculent phenotype. Furthermore, deletion of HOG1 led to

  15. The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger

    PubMed Central

    Priegnitz, Bert-Ewald; Brandt, Ulrike; Pahirulzaman, Khomaizon A. K.; Dickschat, Jeroen S.

    2015-01-01

    Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi. PMID:25888553

  16. The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger.

    PubMed

    Priegnitz, Bert-Ewald; Brandt, Ulrike; Pahirulzaman, Khomaizon A K; Dickschat, Jeroen S; Fleißner, André

    2015-06-01

    Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi. PMID:25888553

  17. The p38α mitogen-activated protein kinase is a key regulator of myelination and remyelination in the CNS.

    PubMed

    Chung, S-H; Biswas, S; Selvaraj, V; Liu, X-B; Sohn, J; Jiang, P; Chen, C; Chmilewsky, F; Marzban, H; Horiuchi, M; Pleasure, D E; Deng, W

    2015-01-01

    The p38α mitogen-activated protein kinase (MAPK) is one of the serine/threonine kinases regulating a variety of biological processes, including cell-type specification, differentiation and migration. Previous in vitro studies using pharmacological inhibitors suggested that p38 MAPK is essential for oligodendrocyte (OL) differentiation and myelination. To investigate the specific roles of p38α MAPK in OL development and myelination in vivo, we generated p38α conditional knockout (CKO) mice under the PLP and nerve/glial antigen 2 (NG2) gene promoters, as these genes are specifically expressed in OL progenitor cells (OPCs). Our data revealed that myelin synthesis was completely inhibited in OLs differentiated from primary OPC cultures derived from the NG2 Cre-p38α CKO mouse brains. Although an in vivo myelination defect was not obvious after gross examination of these mice, electron microscopic analysis showed that the ultrastructure of myelin bundles was severely impaired. Moreover, the onset of myelination in the corpus callosum was delayed in the knockout mice compared with p38α fl/fl control mice. A delay in OL differentiation in the central nervous system was observed with concomitant downregulation in the expression of OPC- and OL-specific genes such as Olig1 and Zfp488 during early postnatal development. OPC proliferation was not affected during this time. These data indicate that p38α is a positive regulator of OL differentiation and myelination. Unexpectedly, we observed an opposite effect of p38α on remyelination in the cuprizone-induced demyelination model. The p38α CKO mice exhibited better remyelination capability compared with p38α fl/fl mice following demyelination. The opposing roles of p38α in myelination and remyelination could be due to a strong anti-inflammatory effect of p38α or a dual reciprocal regulatory action of p38α on myelin formation during development and on remyelination after demyelination. PMID:25950478

  18. A mitogen-activated protein kinase kinase inhibitor induced compound skin toxicity with oedema in metastatic malignant melanoma.

    PubMed

    Thomas, C L; Mortimer, P S; Larkin, J M; Basu, T N; Gore, M E; Fearfield, L

    2016-04-01

    We report three cases of skin toxicity associated with oral mitogen-activated protein kinase kinase (MEK) inhibitor treatment for metastatic malignant melanoma (MM). All three patients developed oedema, and a single patient experienced eyelash trichomegaly. This is the first known report of eyelash trichomegaly secondary to MEK inhibitor use. We also discuss possible mechanisms for MEK inhibitor-associated oedema development. This series supports the role of the dermatologist in the screening and management of patients in the rapidly developing oncology setting, as new targeted agents can give rise to marked skin toxicity. PMID:26411345

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

    PubMed

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

    2002-07-01

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

  20. Selective response of ternary complex factor Sap1a to different mitogen-activated protein kinase subgroups.

    PubMed Central

    Strahl, T; Gille, H; Shaw, P E

    1996-01-01

    Mitogenic and stres signals results in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases. A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1, which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals. A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which is not clear. Here we show that Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs. Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs. Moreover, Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress. These results suggest that Sap1a and Elk-1 have distinct physiological functions. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8876175

  1. Detection of phosphorylated mitogen-activated protein kinase in the developing spinal cord of the mouse embryo

    SciTech Connect

    Teraishi, Toshiya; Miura, Kenji

    2011-09-16

    Highlights: {yields} We detected physiologically phosphorylated MAPKs in developing spinal cord. {yields} We detected physiologically phosphorylated MAPKs by an improved method. {yields} p-ERK1/2 and p-JNK1/2 were detected in the marginal layer and the dorsal horn. {yields} p-ERK1/2 and p-JNK1/2 might play critical roles in the developing spinal cord. {yields} Constructing phosphoprotein atlases will be possible if expanding this work. -- Abstract: Global understanding of the proteome is a major research topic. The comprehensive visualization of the distribution of proteins in vivo or the construction of in situ protein atlases may be a valuable strategy for proteomic researchers. Information about the distribution of various proteins under physiological and pathological conditions should be extremely valuable for the basic and clinical sciences. The mitogen-activated protein kinase (MAPK) cascade plays an essential role in intracellular signaling in organisms. This cascade also regulates biological processes involving development, differentiation, and proliferation. Phosphorylation and dephosphorylation are integral reactions in regulating the activity of MAPKs. Changes in the phosphorylation state of MAPKs are rapid and reversible; therefore, the localizations of physiologically phosphorylated MAPKs in vivo are difficult to accurately detect. Furthermore, phosphorylated MAPKs are likely to change phosphorylated states through commonly used experimental manipulations. In the present study, as a step toward the construction of in situ phosphoprotein atlases, we attempted to detect physiologically phosphorylated MAPKs in vivo in developing spinal cords of mice. We previously reported an improved immunohistochemical method for detecting unstable phosphorylated MAPKs. The distribution patterns of phosphorylated MAPKs in the spinal cords of embryonic mice from embryonic day 13 (E13) to E17 were observed with an improved immunohistochemical method. Phosphorylated

  2. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways.

    PubMed

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  3. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways

    PubMed Central

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K.; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  4. Treponema denticola Activates Mitogen-Activated Protein Kinase Signal Pathways through Toll-Like Receptor 2▿

    PubMed Central

    Ruby, John; Rehani, Kunal; Martin, Michael

    2007-01-01

    Treponema denticola, a spirochete indigenous to the oral cavity, is associated with host inflammatory responses to anaerobic polymicrobial infections of the root canal, periodontium, and alveolar bone. However, the cellular mechanisms responsible for the recognition of T. denticola by the innate immune system and the underlying cell signaling pathways that regulate the inflammatory response to T. denticola are currently unresolved. In this study, we demonstrate that T. denticola induces innate immune responses via the utilization of Toll-like receptor 2 (TLR2) but not TLR4. Assessment of TLR2/1 and TLR2/6 heterodimers revealed that T. denticola predominantly utilizes TLR2/6 for the induction of cellular responses. Analysis of the mitogen-activated protein kinase (MAPK) signaling pathway in T. denticola-stimulated monocytes identified a prolonged up-regulation of the MAPK extracellular signal-related kinase 1/2 (ERK1/2) and p38, while no discernible increase in phospho-c-Jun N-terminal kinase 1/2 (JNK1/2) levels was observed. With the aid of pharmacological inhibitors selectively targeting ERK1/2 via the mitogen-activated protein kinase/extracellular signal-related kinase 1/2 kinase and p38, we further demonstrate that ERK1/2 and p38 play a major role in T. denticola-mediated pro- and anti-inflammatory cytokine production. PMID:17923521

  5. Contractions Activate Hormone-Sensitive Lipase in Rat Muscle by Protein Kinase C and Mitogen-Activated Protein Kinase

    PubMed Central

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia; Ploug, Thorkil; Galbo, Henrik

    2003-01-01

    Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50 % by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant from basal but not from electrically stimulated muscle. In conclusion, in muscle, PKC can stimulate HSL through ERK. Contractions and adrenaline enhance muscle HSL activity by different signalling mechanisms. The effect of contractions is mediated by PKC, at least partly via the ERK pathway. PMID:12794177

  6. Mitogen-activated protein kinases in the porcine retinal arteries and neuroretina following retinal ischemia-reperfusion

    PubMed Central

    Gesslein, Bodil; Håkansson, Gisela; Carpio, Ronald; Gustafsson, Lotta; Perez, Maria-Thereza

    2010-01-01

    Purpose The aim of the present study was to examine changes in the expression of intracellular signal-transduction pathways, specifically mitogen-activated protein kinases, following retinal ischemia-reperfusion. Methods Retinal ischemia was induced by elevating the intraocular pressure in porcine eyes, followed by 5, 12, or 20 h of reperfusion. The results were compared to those of the sham- operated fellow eye. The retinal arteries and neuroretina were isolated separately and examined. Tissue morphology and DNA fragmentation were studied using histology. Extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, c-junNH2-terminal kinases (JNK), and c-jun protein and mRNA expression were examined using immunofluorescence staining, western blot, and real-time PCR techniques. Results Pyknotic cell nuclei, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, and glial fibrillary acidic protein mRNA expression were increased in ischemia, suggesting injury. Phosphorylated ERK1/2 protein levels were increased in the neuroretina following ischemia, while mRNA levels were unaltered. p38 protein and mRNA levels were not affected by ischemia. Immunofluorescence staining for phosphorylated p38 was especially intense in the retinal blood vessels, while only weak in the neuroretina. Phosphorylated JNK protein and mRNA were slightly decreased in ischemia. Phosphorylated c-jun protein and mRNA levels were higher in the neuroretina after ischemia-reperfusion. Conclusions Retinal ischemia-reperfusion alters expression of mitogen-activated protein kinases, particularly ERK1/2, in the neuroretina and retinal arteries. The development of pharmacological treatment targeting these intracellular transduction pathways may prevent injury to the eye following retinal circulatory failure. PMID:20300568

  7. Epstein-Barr virus latent membrane protein 2 associates with and is a substrate for mitogen-activated protein kinase.

    PubMed

    Panousis, C G; Rowe, D T

    1997-06-01

    The latent membrane protein 2 (LMP2) of Epstein-Barr virus interferes with B-lymphocyte signal transduction through the immunoglobulin (Ig) receptor. Two isoforms of LMP2 exist and differ only in that one isoform (LMP2a) contains an N-terminal cytoplasmic domain that the other isoform does not. LMP2a is a phosphoprotein that is phosphorylated on tyrosines and serines in the cytoplasmic domain. GST1-119, a glutathione S-transferase (GST) fusion protein containing the 119 amino acids of the cytoplasmic domain, affinity precipitated serine kinase activity from BJAB cell extracts. The affinity-precipitated kinase phosphorylated LMP2a sequences, and kinase activity was increased following induction. Probing of Western immunoblots of affinity-precipitated proteins showed that the Erk1 form of mitogen-activated protein kinase (MAPK) was present. Purified MAPK phosphorylated GST fusion proteins containing the cytoplasmic domain of LMP2a and mutational analyses were used to identify S15 and S102 as the sites of in vitro phosphorylation. A polyclonal rabbit antiserum was prepared against a maltose binding protein-LMP2a cytoplasmic domain fusion protein (MBP1-119) and used to immunoprecipitate LMP2a from the in vitro-immortalized lymphoblastoid B-cell line B95-8CR. LMP2a immunoprecipitates from B95-8CR contained MAPK as a coprecipitated protein. Cross-linking surface Ig on B95-8CR cells failed to induce MAPK activity within the cells. Treatment of B95-8CR with phorbol myristate acetate (PMA) was able to bypass the Ig receptor block and activate MAPK activity. Phosphorylation of LMP2a on serine residues increased after PMA induction. The possible role for LMP2a serine phosphorylation by MAPK in the control of latency is discussed. PMID:9151869

  8. Quercetin attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways.

    PubMed

    Li, Chengqiu; Wang, Ting; Zhang, Chunyuan; Xuan, Jichang; Su, Changjiang; Wang, Yuqi

    2016-02-15

    Quercetin (Que), a plant-derived flavonoid, possesses various biological functions. Moreover, Que exerts multiple beneficial actions in treatment of cardiovascular diseases and there are an inverse association between Que intakes and occurrence and development of various cardiovascular diseases. Some researchers have inferred that the mechanisms of Que to protect cardiomyocytes from ischemia/reperfusion (I/R) injury may be involved in modulation of intracellular signal pathways and regulation of proteins expression in vivo. The current study investigated whether Que has any protective effects on cardiomyocytes from hypoxia/reoxygenation (H/R) in vitro and its potential cardioprotective mechanisms. The cell viability of Que on H9c2 cardiomyoblast cells was assessed by MTT. Apoptosis was evaluated by both Hoechst33342 staining and Flow cytometric analysis (FACS). Furthermore, the effect of Que, SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor) on mitogen-activated protein kinases (MAPKs) and the expression of apoptosis related proteins (Bcl-2, Bax and caspase-3) was determined by Western blotting. MTT assays showed that pretreatment with Que could increase the viability of H9c2 cardiomyocytes that suffered H/R. Both Hoechst33342 staining and FACS confirmed that Que could remarkably suppress the H/R-induced apoptotic cardiomyocytes. In addition, Que significantly alleviated H/R-induced the phosphorylation of JNK and p38, which further increased Bcl-2 expression and inhibited the activation of Bax and caspase-3 directly or indirectly. In summary, our results imply that Que can induce cardioprotection by inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways and modulate the expression of Bcl-2 and Bax proteins that provides a new experimental foundation for myocardial ischemia disease therapy. PMID:26680104

  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. Differential activation of mitogen-activated protein kinases following high and low LET radiation in murine macrophage cell line.

    PubMed

    Narang, Himanshi; Bhat, Nagesh; Gupta, S K; Santra, S; Choudhary, R K; Kailash, S; Krishna, Malini

    2009-04-01

    Mitogen-activated protein kinases have been shown to respond to various stimuli including cytokines, mitogens and gamma irradiation, leading to cell proliferation, differentiation, or death. The duration of their activation determines the specificity of response to each stimulus in various cells. In this study, the crucial intracellular kinases, ERK, JNK, and p38 kinase involved in cell survival, death, or damage and repair were examined for their activity in RAW 264.7 cells at various time points after irradiation with 2 Gy doses of proton ions or X-rays. This is the first report that shows that the MAPK signaling induced after heavy ion or X-ray exposure is not the same. Unlike gamma irradiation, there was prolonged but marginal activation of prosurvival ERK pathway and significant activation of proapoptotic p38 pathway in response to high LET radiation. PMID:19112558

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

    PubMed

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

    1996-12-24

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

  12. Botulinum toxin complex increases paracellular permeability in intestinal epithelial cells via activation of p38 mitogen-activated protein kinase.

    PubMed

    Miyashita, Shin-Ichiro; Sagane, Yoshimasa; Inui, Ken; Hayashi, Shintaro; Miyata, Keita; Suzuki, Tomonori; Ohyama, Tohru; Watanabe, Toshihiro; Niwa, Koichi

    2013-12-30

    Clostridium botulinum produces a large toxin complex (L-TC) that increases paracellular permeability in intestinal epithelial cells by a mechanism that remains unclear. Here, we show that mitogen-activated protein kinases (MAPKs) are involved in this permeability increase. Paracellular permeability was measured by FITC-dextran flux through a monolayer of rat intestinal epithelial IEC-6 cells, and MAPK activation was estimated from western blots. L-TC of C. botulinum serotype D strain 4947 increased paracellular dextran flux and activated extracellular signal-regulated kinase (ERK), p38, but not c-Jun N-terminal kinase (JNK) in IEC-6 cells. The permeability increase induced by L-TC was abrogated by the p38 inhibitor SB203580. These results indicate that L-TC increases paracellular permeability by activating p38, but not JNK and ERK. PMID:23884081

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

    PubMed

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

    2016-07-29

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

  14. 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

    PubMed Central

    Sithanandam, G; Latif, F; Duh, F M; Bernal, R; Smola, U; Li, H; Kuzmin, I; Wixler, V; Geil, L; Shrestha, S

    1996-01-01

    NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity. PMID:8622688

  15. p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion

    PubMed Central

    George, Margaret D.; Wine, Robert N.; Lackford, Brad; Kissling, Grace E.; Akiyama, Steven K.; Olden, Kenneth; Roberts, John D.

    2014-01-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading and that this association can be regulated by factors in the tumor microenvironment. PMID:24219282

  16. Studies on mitogen-activated protein kinase signaling pathway in the alveolar macrophages of chronic bronchitis rats.

    PubMed

    Huang, Yan; Meng, Xiao-Ming; Jiang, Guo-Lin; Yang, Ya-Ru; Liu, Juan; Lv, Xiong-Wen; Li, Jun

    2015-02-01

    Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in alveolar macrophages (AMs), activates several intracellular signaling pathways, including mitogen-activated protein kinases (MAPK). In the present study, we investigated the MAPK pathway in AMs of chronic bronchitis (CB) rats. CB was induced by endotracheal instillation of LPS followed by Bacillus Calmette Guerin injection through the caudal vein 1 week later. Specific inhibitors were used and protein phosphorylations were detected by Western blot. We found that Genistein (PTK inhibitor) could inhibit protein kinase C (PKC), phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt or PKB) MAPK signaling pathway with different degrees, LY294002 (PI3K inhibitor) could not only inhibit phospho-PI3K/Akt expression, but also inhibit p38 and c-Jun NH2-terminal kinases (JNK) phosphorylation. Calphostin C (PKC inhibitor) could inhibit phospho-PKC expression and exerted significant effects on extracellular signal-regulated kinases (ERK) phosphorylation, however, it had no impact on p38 and JNK phosphorylation. These results demonstrated that the LPS mediated signaling pathway of MAPK in AMs of CB rats could be described as follows: PTK-PI3K-Akt-JNK/p38 or PTK-PI3K-PKC-ERK, and PI3K may have a negative regulation on the activation of downstream proteins. PMID:25467375

  17. 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...

  18. Postextinction Infusion of a Mitogen-Activated Protein Kinase Inhibitor into the Medial Prefrontal Cortex Impairs Memory of the Extinction of Conditioned Fear

    ERIC Educational Resources Information Center

    Hugues, Sandrine; Deschaux, Olivier; Garcia, Rene

    2004-01-01

    We investigated whether postextinction training infusion of PD098059, a selective inhibitor of mitogen-activated protein kinase (MAPK) activation, into the medial prefrontal cortex, would impair retention of extinction learning in rats. We found that immediate, but not late (2 or 4 h), postextinction infusion of PD098059 provoked a full return of…

  19. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava

    PubMed Central

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars. PMID:27625666

  20. Allopurinol induces innate immune responses through mitogen-activated protein kinase signaling pathways in HL-60 cells.

    PubMed

    Nakajima, Akira; Oda, Shingo; Yokoi, Tsuyoshi

    2016-09-01

    Allopurinol, an inhibitor of xanthine oxidase, is a frequent cause of severe cutaneous adverse reactions (SCARs) in humans, including drug rash with eosinophilia and systemic symptoms, Stevens-Johnson syndrome and toxic epidermal necrolysis. Although SCARs have been suspected to be immune-mediated, the mechanisms of allopurinol-induced SCARs remain unclear. In this study, we examined whether allopurinol has the ability to induce innate immune responses in vitro using human dendritic cell (DC)-like cell lines, including HL-60, THP-1 and K562, and a human keratinocyte cell line, HaCaT. In this study, we demonstrate that treatment of HL-60 cells with allopurinol significantly increased the mRNA expression levels of interleukin-8, monocyte chemotactic protein-1 and tumor necrosis factor α in a time- and concentration-dependent manner. Furthermore, allopurinol induced the phosphorylation of mitogen-activated protein kinases (MAPK), such as c-Jun N-terminal kinase and extracellular signal-regulated kinase, which regulate cytokine production in DC. In addition, allopurinol-induced increases in cytokine expression were inhibited by co-treatment with the MAPK inhibitors. Collectively, these results suggest that allopurinol has the ability to induce innate immune responses in a DC-like cell line through activation of the MAPK signaling pathways. These results indicate that innate immune responses induced by allopurinol might be involved in the development of allopurinol-induced SCARs. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26641773

  1. Jasmonate-based wound signal transduction requires activation of WIPK, a tobacco mitogen-activated protein kinase.

    PubMed Central

    Seo, S; Sano, H; Ohashi, Y

    1999-01-01

    A gene encoding a tobacco mitogen-activated protein kinase (WIPK) is transcriptionally activated in response to wounding. Transgenic tobacco plants, in which expression of endogenous wipk was suppressed, did not accumulate jasmonic acid or its methyl ester when wounded, suggesting that WIPK is involved in jasmonate-mediated wound signal transduction. Here, we demonstrate that activation of WIPK is required for triggering the jasmonate-mediated signal transduction cascade that occurs when wild-type tobacco plants are wounded. We also show that when plants are wounded, WIPK is rapidly and transiently activated, whereas the quantity of WIPK protein is maintained at a constant level. A transgenic tobacco plant in which the wipk gene was constitutively expressed at a high level showed constitutive enzymatic activation of WIPK and exhibited three- to fourfold higher levels of jasmonate than did its wild-type counterpart. This plant also showed constitutive accumulation of jasmonate-inducible proteinase inhibitor II transcripts. These results show that WIPK is activated in response to wounding, which subsequently causes an increase in jasmonate synthesis. PMID:9927645

  2. Mitogen-activated protein kinase cascade required for regulation of development and secondary metabolism in Neurospora crassa.

    PubMed

    Park, Gyungsoon; Pan, Songqin; Borkovich, Katherine A

    2008-12-01

    Mitogen-activated protein kinase (MAPK) signaling cascades are composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In this study, we characterize components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; and mak-1, MAPK) homologous to that controlling cell wall integrity in Saccharomyces cerevisiae. Growth of basal hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. All three mutants formed short aerial hyphae and the formation of asexual macroconidia was reduced in Deltamik-1 mutants and almost abolished in Deltamek-1 and Deltamak-1 strains. In contrast, the normally rare asexual spores, arthroconidia, were abundant in cultures of the three mutants. Deltamik-1, Deltamek-1, and Deltamak-1 mutants were unable to form protoperithecia or perithecia when used as females in a sexual cross. The MAK-1 MAPK was not phosphorylated in Deltamik-1 and Deltamek-1 mutants, consistent with the involvement of MIK-1, MEK-1, and MAK-1 in the same signaling cascade. Interestingly, we observed increased levels of mRNA and protein for tyrosinase in the mutants under nitrogen starvation, a condition favoring sexual differentiation. Tyrosinase is an enzyme that catalyzes production of the secondary metabolite l-DOPA melanin. These results implicate the MAK-1 pathway in regulation of development and secondary metabolism in filamentous fungi. PMID:18849472

  3. Induction of p38 mitogen-activated protein kinase reduces early endosome autoantigen 1 (EEA1) recruitment to phagosomal membranes.

    PubMed

    Fratti, Rutilio A; Chua, Jennifer; Deretic, Vojo

    2003-11-21

    Mycobacterium tuberculosis survives in the infected host by parasitizing macrophages in which the bacillus resides in a specialized phagosome sequestered from the phagolysosomal degradative pathway. Here we report a role of the stress-induced p38 mitogen-activated protein kinase (p38 MAPK) in the component of M. tuberculosis phagosome maturation arrest that has been linked previously to the reduced recruitment of the endosomal and phagosomal membrane-tethering molecule called early endosome autoantigen 1 (EEA1; Fratti, R. A., Backer, J. M., Gruenberg, J., Corvera, S., and Deretic, V. (2001) J. Cell Biol. 154, 631-644). A pharmacological inhibition of M. tuberculosis var. bovis Bacillus Calmette-Guérin-induced p38 MAPK activity caused a marked increase in EEA1 colocalization with mycobacterial phagosomes. Consistent with the increase in EEA1 association and its role in phagosomal maturation, the pharmacological block of p38 activity caused phagosomal acidification and enrichment of the late endocytic markers lysobisphosphatidic acid and CD63 (lysosomal integral membrane protein 1) on mycobacterial phagosomes. A negative regulatory role of p38 MAPK activation in phagosome maturation was further demonstrated by converse experiments with latex bead phagosomes. Artificial activation of p38 MAPK caused a decrease in EEA1 colocalization with model latex bead phagosomes, which normally acquire EEA1 and subsequently mature into the phagolysosome. These findings show that p38 MAPK activity contributes to the arrest of M. tuberculosis phagosome maturation and demonstrate a negative regulatory role of p38 in phagolysosome biogenesis. PMID:12963735

  4. Adult-onset hyperthyroidism impairs spatial learning: possible involvement of mitogen-activated protein kinase signaling pathways.

    PubMed

    Bitiktaş, Soner; Kandemir, Başak; Tan, Burak; Kavraal, Şehrazat; Liman, Narin; Dursun, Nurcan; Dönmez-Altuntaş, Hamiyet; Aksan-Kurnaz, Işil; Suer, Cem

    2016-08-01

    Given evidence that mitogen-activated protein kinase (MAPK) activation is part of the nongenomic actions of thyroid hormones, we investigated the possible consequences of hyperthyroidism for the cognitive functioning of adult rats. Young adult rats were treated with L-thyroxine or saline. Twenty rats in each group were exposed to Morris water maze testing, measuring their performance in a hidden-platform spatial task. In a separate set of rats not exposed to Morris water maze testing (untrained rats), the expression and phosphorylated levels of p38-MAPK and of its two downstream effectors, Elk-1 and cAMP response element-binding protein, were evaluated using quantitative reverse transcriptase-PCR and western blotting. Rats with hyperthyroidism showed delayed acquisition of learning compared with their wild-type counterparts, as shown by increased escape latencies and distance moved on the last two trials of daily training in the water maze. The hyperthyroid rats, however, showed no difference during probe trials. Western blot analyses of the hippocampus showed that hyperthyroidism increased phosphorylated p38-MAPK levels in untrained rats. Although our study is correlative in nature and does not exclude the contribution of other molecular targets, our findings suggest that the observed impairments in acquisition during actual learning in rats with hyperthyroidism may result from the increased phosphorylation of p38-MAPK. PMID:27258653

  5. Differential activation by cytokines of mitogen-activated protein kinases in bovine temporomandibular-joint disc cells.

    PubMed

    Landesberg, R; Takeuchi, E; Puzas, J E

    1999-01-01

    Temporomandibular disorders affect a significant proportion of the population. While their aetiology is not well defined, recent histological studies suggest that the majority are similar to the osteoarthritis seen in other joints. Inflammatory cytokines such as interleukin-1 and tumour necrosis factor-alpha appear to be important in the cascade of events leading to joint destruction in osteoarthritis. Here, cells from the disc of bovine temporomandibular joint were used to examine the response to various cytokines in vitro. Disc cells were stimulated with interleukin-1alpha, tumour necrosis factor-alpha, transforming growth factor-beta, platelet-derived growth factor, and basic fibroblast growth factor. Their effects were monitored by assessing the phosphorylation of selected signal-transduction intermediates using western blot. Mitogen-activated protein kinases (Erk 1, Erk 2) were rapidly phosphorylated by exposure to basic fibroblast growth factor, platelet-derived growth factor, and tumour necrosis factor-alpha, while interleukin-1alpha showed a weak response. Transforming growth factor-beta failed to activate these kinases. Examination of the effect of these cytokines on p38 (an intermediate in the stress-activated protein-kinase pathway) showed an increase in phosphorylated p38 when stimulated with tumour necrosis factor-alpha and interleukin-1alpha. The amounts of phosphorylated signal transducer and activator of transcription-3 did not significantly increase when the cells were exposed to any of the cytokines. PMID:10075149

  6. Ethanol alters angiotensin II stimulated mitogen activated protein kinase in hepatocytes: agonist selectivity and ethanol metabolic independence.

    PubMed

    Weng, Y; Shukla, S D

    2000-06-23

    Angiotensin II activated mitogen-activated protein kinase (MAPK) (p42 and p44) in rat hepatocytes exposed to ethanol and the relevance of ethanol metabolism on this activation was investigated. Hepatocytes, isolated from rat liver, were treated with or without ethanol for 24 h. Angiotensin II, vasopressin, insulin, serum and epinephrine significantly increased hepatocyte MAPK activity. Platelet activating factor (PAF), tumor necrosis factor-alpha (TNF-alpha), and insulin-like growth factor-1 (IGF-1) had little effect on MAPK activation. Interestingly, among the above agonists, which activated hepatocyte MAPK, ethanol exposure potentiated only angiotensin II and epinephrine-stimulated MAPK. Thus, potentiation of MAPK by ethanol exhibited agonist selectivity. In contrast to several other cells, there was prevalence of p42 over p44 MAPK band in hepatocytes. Angiotensin II treatment caused a rapid activation (peak 5 min) of MAPK followed by a decrease to basal levels in 30 min. Exposure with 100 mM ethanol potentiated the angiotensin II stimulated MAPK activity. This potentiation was partially blocked by pertussis toxin suggesting it to be a G-protein-dependent event. Treatment of the hepatocytes with pyrazole (an inhibitor of ethanol metabolism) or acetaldehyde (an ethanol metabolite) had no effect on potentiation. Thus, ethanol potentiation of hepatocyte MAPK is agonist-selective and independent of ethanol metabolism. PMID:10862821

  7. Analysis of Mitogen-Activated Protein Kinases in Bone and Cartilage of Patients with Rheumatoid Arthritis Treated with Abatacept

    PubMed Central

    Kanbe, Katsuaki; Oh, Koei; Chiba, Junji; Inoue, Yasuo; Taguchi, Masashi; Yabuki, Akiko

    2016-01-01

    The aim of this study was to analyze the histological changes related to mitogen-activated protein (MAP) kinases in bone and cartilage treated with abatacept for rheumatoid arthritis (RA). A total of 20 patients of bone and cartilage were assessed: 10 abatacept with methotrexate (MTX)-treated RA patients were compared with 10 MTX-treated RA patients (control). The histology of bone and cartilage was observed by staining with hematoxylin and eosin and analyzed immunohistochemically for the expression of tumor necrosis factor-α, interleukin-6, CD4 (T cell), CD68 (macrophage), receptor activator of nuclear kappa-B ligand, osteoprotegerin, osteopontin, CD29 (β-1 integrin), phospho-p38 MAPK (Tyr180/Tyr182), phospho-p44/42 MAPK (extracellular signal-regulated kinase, ERK1/ERK2), and phosphor-c-Jun N-terminal kinase. The expressions of CD29 known as mechanoreceptor and ERK known as mechanotransduction signal protein in MAP kinases in the bone and cartilage of patients treated with abatacept were significantly different from those of control. These findings suggest that increases in CD29 and ERK in MAP kinases may change the metabolism of bone and cartilage in RA patients treated with abatacept. PMID:27103846

  8. Mitogen-activated protein kinase p38b interaction with delta class glutathione transferases from the fruit fly, Drosophila melanogaster.

    PubMed

    Wongtrakul, Jeerang; Sukittikul, Suchada; Saisawang, Chonticha; Ketterman, Albert J

    2012-01-01

    Glutathione transferases (GSTs) are a family of multifunctional enzymes involved in xenobiotic biotransformation, drug metabolism, and protection against oxidative damage. The p38b mitogen-activated protein kinase is involved in cellular stress response. This study screened interactions between Drosophila melanogaster Meigen (Diptera: Drosophilidae) Delta class glutathione transferases (DmGSTs) and the D. melanogaster p38b MAPK. Therefore, 12 DmGSTs and p38b kinase were obtained as recombinant proteins. The study showed that DmGSTD8 and DmGSTD11b significantly increased p38b activity toward ATF2 and jun, which are transcription factor substrates. DmGSTD3 and DmGSTD5 moderately increased p38b activity for jun. In addition, GST activity in the presence of p38b was also measured. It was found that p38b affected substrate specificity toward CDNB (1-chloro-2,4-dinitrobenzene) and DCNB (1,2-dichloro-4-nitrobenzene) of several GST isoforms, i.e., DmGSTD2, DmGSTD5, DmGSTD8, and DmGSTD11b. The interaction of a GST and p38b can affect the substrate specificity of either enzyme, which suggests induced conformational changes affecting catalysis. Similar interactions do not occur for all the Delta enzymes and p38b, which suggests that these interactions could be specific. PMID:23438069

  9. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava.

    PubMed

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars. PMID:27625666

  10. Liquiritigenin Induces Tumor Cell Death through Mitogen-Activated Protein Kinase- (MPAKs-) Mediated Pathway in Hepatocellular Carcinoma Cells

    PubMed Central

    Lu, Jiahui; Liu, Yan; Meng, Qingfan; Xie, Jing; Wang, Zhenzuo

    2014-01-01

    Liquiritigenin (LQ), separated from Glycyrrhiza radix, possesses anti-inflammatory, antihyperlipidemic, and antiallergic effects. Our present study aims to investigate the antihepatocellular carcinoma effects of LQ both in cell and animal models. LQ strikingly reduced cell viability, enhanced apoptotic rate, induced lactate dehydrogenase over-release, and increased intracellular reactive oxygen species (ROS) level and caspase 3 activity in both PLC/PRL/5 and HepG2 cells. The expression of cleaved PARP, the hall-marker of apoptosis, was enhanced by LQ. LQ treatment resulted in a reduction of the expressions of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL), and an increase of the phosphorylation of c-Jun N-terminal kinases (JNK) and P38. LQ-mediated cell viability reduction, mitochondrial dysfunction, apoptosis related protein abnormal expressions, and JNK and P38 activation were partially abolished by N-Acetyl-L-cysteine (a ROS inhibitor) pretreatment. Moreover, LQ suppressed the activation of extracellular signaling-regulated kinase (ERKs) and reduced the translocation of phosphor-ERKs from cytoplasm to nucleus. This antitumor activity was further confirmed in PLC/PRL/5-xenografted mice model. All these data indicate that the antihepatocellular carcinoma effects of LQ are related to its modulation of the activations of mitogen-activated protein kinase (MAPKs). The study provides experimental evidence supporting LQ as a potential therapeutic agent for hepatocellular carcinoma treatment. PMID:24738081

  11. Human immunodeficiency virus type 1 Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity.

    PubMed Central

    Greenway, A; Azad, A; Mills, J; McPhee, D

    1996-01-01

    It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis. PMID:8794306

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

    SciTech Connect

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

    2012-03-02

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

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

    PubMed

    Takezawa, D

    1999-08-01

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

  14. Analysis of mitogen-activated protein kinase pathways used by interleukin 1 in tissues in vivo: activation of hepatic c-Jun N-terminal kinases 1 and 2, and mitogen-activated protein kinase kinases 4 and 7.

    PubMed Central

    Finch, A; Davis, W; Carter, W G; Saklatvala, J

    2001-01-01

    The effects of interleukin 1 (IL-1) are mediated by the activation of protein kinase signalling pathways, which have been well characterized in cultured cells. We have investigated the activation of these pathways in rabbit liver and other tissues after the systemic administration of IL-1alpha. In liver there was 30-40-fold activation of c-Jun N-terminal kinase (JNK) and 5-fold activation of both JNK kinases, mitogen-activated protein kinase (MAPK) kinase (MKK)4 and MKK7. IL-1alpha also caused 2-3-fold activation of p38 MAPK and degradation of the inhibitor of nuclear factor kappaB ('IkappaB'), although no activation of extracellular signal-regulated protein kinase (ERK) (p42/44 MAPK) was observed. The use of antibodies against specific JNK isoforms showed that, in liver, short (p46) JNK1 and long (p54) JNK2 are the predominant forms activated, with smaller amounts of long JNK1 and short JNK2. No active JNK3 was detected. A similar pattern of JNK activation was seen in lung, spleen, skeletal muscle and kidney. Significant JNK3 activity was detectable only in the brain, although little activation of the JNK pathway in response to IL-1alpha was observed in this tissue. This distribution of active JNK isoforms probably results from a different expression of JNKs within the tissues, rather than from a selective activation of isoforms. We conclude that IL-1alpha might activate a more restricted set of signalling pathways in tissues in vivo than it does in cultured cells, where ERK and JNK3 activation are often observed. Cultured cells might represent a 'repair' phenotype that undergoes a broader set of responses to the cytokine. PMID:11139391

  15. Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase

    NASA Astrophysics Data System (ADS)

    Koo, Han-Mo; Vanbrocklin, Matt; McWilliams, Mary Jane; Leppla, Stephan H.; Duesbery, Nicholas S.; Vande Woude, George F.

    2002-03-01

    Lethal factor, the principal virulence factor of Bacillus anthracis, inhibits mitogen-activated protein kinase (MAPK) signaling by proteolytically cleaving MAPK kinases. Edema factor, another component of anthrax toxin, is an adenylate cyclase, which increases intracellular cAMP. Inhibition of MAPK signaling with either anthrax lethal toxin (LeTx) or small molecule MAPK kinase inhibitors triggers apoptosis in human melanoma cells. Normal melanocytes do not undergo apoptosis in response to MAPK inhibition but arrest in the G1 phase of the cell cycle. Importantly, in vivo treatment of human melanoma xenograft tumors in athymic nude mice with LeTx results in significant or complete tumor regression without apparent side effects, suggesting that inhibiting the MAPK signaling pathway may be a useful strategy for treating melanoma. Additionally, interrupting MAPK signaling with LeTx and elevating cAMP with anthrax edema toxin in both melanoma cells and melanocytes lead to dramatic melanin production, perhaps explaining the formation of blackened eschars in cutaneous anthrax.

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

    PubMed

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

    2012-06-01

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

  17. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

    PubMed Central

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M.

    2010-01-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets. PMID:19965619

  18. Interplay between mitogen-activated protein kinase and nitric oxide in brassinosteroid-induced pesticide metabolism in Solanum lycopersicum.

    PubMed

    Yin, Yan-Ling; Zhou, Yue; Zhou, Yan-Hong; Shi, Kai; Zhou, Jie; Yu, Yunlong; Yu, Jing-Quan; Xia, Xiao-Jian

    2016-10-01

    Nitric oxide (NO) and mitogen-activated protein kinase (MPK) play important roles in brassinosteroid (BR)-induced stress tolerance, however, their functions in BR-induced pesticides metabolism remain unclear. Here, we showed that MPK activity and transcripts of SlMPK1 and SlMPK2 were induced by chlorothalonil (CHT), a widely used fungicide, in tomato leaves. However, cosilencing of SlMPK1/2 compromised the 24-epibrassinolide (EBR)-induced upregulation of detoxification genes and CHT metabolism in tomato leaves. In addition, cosilencing of SlMPK1/2 inhibited the accumulation of S-nitrosothiol (SNO), the reservoir of nitric oxide (NO) in plants, whereas tungstate, the inhibitor of nitrate reductase (NR), blocked EBR-induced SNO accumulation and MPK activity. Inhibiting the accumulation of NO by cPTIO, the specific scavenger and tungstate abolished the EBR-induced upregulation of detoxification genes, glutathione accumulation and CHT metabolism. The results showed that MPK and NR-dependent NO were involved in BR-induced CHT metabolism. Notably, there was a positive crosstalk between the MPK and NO production. PMID:27236431

  19. Mitogen-Activated Protein Kinase Signaling in the Heart: Angels Versus Demons in a Heart-Breaking Tale

    PubMed Central

    ROSE, BETH A.; FORCE, THOMAS; WANG, YIBIN

    2013-01-01

    Among the myriad of intra-cellular signaling networks that govern the cardiac development and pathogenesis, mitogen-activated protein kinases (MAPKs) are prominent players that have been the focus of extensive investigations in the past decades. The four best characterized MAPK subfamilies, ERK1/2, JNK, p38, and ERK5, are the targets of pharmacological and genetic manipulations to uncover their roles in cardiac development, function, and diseases. However, information reported in the literature from these efforts has not yet resulted in a clear view about the roles of specific MAPK pathways in heart. Rather, controversies from contradictive results have led to a perception that MAPKs are ambiguous characters in heart with both protective and detrimental effects. The primary object of this review is to provide a comprehensive overview of the current progress, in an effort to highlight the areas where consensus is established verses the ones where controversy remains. MAPKs in cardiac development, cardiac hypertrophy, ischemia/reperfusion injury, and pathological remodeling are the main focuses of this review as these represent the most critical issues for evaluating MAPKs as viable targets of therapeutic development. The studies presented in this review will help to reveal the major challenges in the field and the limitations of current approaches and point to a critical need in future studies to gain better understanding of the fundamental mechanisms of MAPK function and regulation in the heart. PMID:20959622

  20. Riboflavin-Induced Disease Resistance Requires the Mitogen-Activated Protein Kinases 3 and 6 in Arabidopsis thaliana.

    PubMed

    Nie, Shengjun; Xu, Huilian

    2016-01-01

    As a resistance elicitor, riboflavin (vitamin B2) protects plants against a wide range of pathogens. At molecular biological levels, it is important to elucidate the signaling pathways underlying the disease resistance induced by riboflavin. Here, riboflavin was tested to induce resistance against virulent Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) in Arabidopsis. Results showed that riboflavin induced disease resistance based on MAPK-dependent priming for the expression of PR1 gene. Riboflavin induced transient expression of PR1 gene. However, following Pst DC3000 inoculation, riboflavin potentiated stronger PR1 gene transcription. Further was suggested that the transcript levels of mitogen-activated protein kinases, MPK3 and MPK6, were primed under riboflavin. Upon infection by Pst DC3000, these two enzymes were more strongly activated. The elevated activation of both MPK3 and MPK6 was responsible for enhanced defense gene expression and resistance after riboflavin treatment. Moreover, riboflavin significantly reduced the transcript levels of MPK3 and MPK6 by application of AsA and BAPTA, an H2O2 scavenger and a calcium (Ca2+) scavenger, respectively. In conclusion, MPK3 and MPK6 were responsible for riboflavin-induced resistance, and played an important role in H2O2- and Ca2+-related signaling pathways, and this study could provide a new insight into the mechanistic study of riboflavin-induced defense responses. PMID:27054585

  1. Prp19 facilitates invasion of hepatocellular carcinoma via p38 mitogen-activated protein kinase/Twist1 pathway

    PubMed Central

    Zhu, Ji-Min; Yu, Qian; Xue, Ru-Yi; Fang, Ying; Zhang, Yi-An; Chen, Yan-Jie; Liu, Tao-Tao; Dong, Ling; Shen, Xi-Zhong

    2016-01-01

    Pre-mRNA processing factor 19 (Prp19) is involved in many cellular events including pre-mRNA processing and DNA damage response. However, the pathological role of Prp19 in hepatocellular carcinoma (HCC) is still elusive. Here, we reported that Prp19 was increased in most HCC tissues and HCC cell lines, and its overexpression in HCC tissues was positively correlated with vascular invasion, tumor capsule breakthrough and poor prognosis. Prp19 potentiated migratory and invasive abilities of HCC cells in vitro and in vivo. Furthermore Prp19 facilitated Twist1-induced epithelial-mesenchymal transition. Mechanistic insights revealed that Prp19 directly binded with TGF-β-activated kinase1 (TAK1) and promoted the activation of p38 mitogen-activated protein kinase (MAPK), preventing Twist1 from degradation. Finally Prp19/p38 MAPK/Twist1 axis was attested in nude mice xenografts and HCC patient specimens. This work implies that the gain of Prp19 is a critical event during the progression of HCC, making it a promising target for malignancies with aberrant Prp19 expression. PMID:26959880

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

    PubMed Central

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

    2015-01-01

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

  3. Riboflavin-Induced Disease Resistance Requires the Mitogen-Activated Protein Kinases 3 and 6 in Arabidopsis thaliana

    PubMed Central

    Nie, Shengjun; Xu, Huilian

    2016-01-01

    As a resistance elicitor, riboflavin (vitamin B2) protects plants against a wide range of pathogens. At molecular biological levels, it is important to elucidate the signaling pathways underlying the disease resistance induced by riboflavin. Here, riboflavin was tested to induce resistance against virulent Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) in Arabidopsis. Results showed that riboflavin induced disease resistance based on MAPK-dependent priming for the expression of PR1 gene. Riboflavin induced transient expression of PR1 gene. However, following Pst DC3000 inoculation, riboflavin potentiated stronger PR1 gene transcription. Further was suggested that the transcript levels of mitogen-activated protein kinases, MPK3 and MPK6, were primed under riboflavin. Upon infection by Pst DC3000, these two enzymes were more strongly activated. The elevated activation of both MPK3 and MPK6 was responsible for enhanced defense gene expression and resistance after riboflavin treatment. Moreover, riboflavin significantly reduced the transcript levels of MPK3 and MPK6 by application of AsA and BAPTA, an H2O2 scavenger and a calcium (Ca2+) scavenger, respectively. In conclusion, MPK3 and MPK6 were responsible for riboflavin-induced resistance, and played an important role in H2O2- and Ca2+-related signaling pathways, and this study could provide a new insight into the mechanistic study of riboflavin-induced defense responses. PMID:27054585

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

    PubMed

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

    2015-08-01

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

  5. The Cotton Mitogen-Activated Protein Kinase Kinase 3 Functions in Drought Tolerance by Regulating Stomatal Responses and Root Growth.

    PubMed

    Wang, Chen; Lu, Wenjing; He, Xiaowen; Wang, Fang; Zhou, Yuli; Guo, Xulei; Guo, Xingqi

    2016-08-01

    Mitogen-activated protein kinase (MAPK) cascades play critical roles in signal transduction processes in eukaryotes. The MAPK kinases (MAPKKs) that link MAPKK kinases (MAPKKKs) and MAPKs are key components of MAPK cascades. However, the intricate regulatory mechanisms that control MAPKKs under drought stress conditions are not fully understood, especially in cotton (Gossypium hirsutum) Here, we isolated and characterized the cotton group B MAPKK gene GhMKK3 Overexpressing GhMKK3 in Nicotiana benthamiana enhanced tolerance to drought, and the results of RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) assays suggest that GhMKK3 plays an important role in responses to abiotic stresses by regulating stomatal responses and root hair growth. Further evidence demonstrated that overexpressing GhMKK3 promoted root growth and ABA-induced stomatal closure. In contrast, silencing GhMKK3 in cotton using virus-induced gene silencing (VIGS) resulted in the opposite phenotypes. More importantly, we identified an ABA- and drought-induced MAPK cascade that is composed of GhMKK3, GhMPK7 and GhPIP1 that compensates for deficiency in the MAPK cascade pathway in cotton under drought stress conditions. Together, these findings significantly improve our understanding of the mechanism by which GhMKK3 positively regulates drought stress responses. PMID:27335349

  6. Signaling via the Trichoderma atroviride mitogen-activated protein kinase Tmk1 differentially affects mycoparasitism and plant protection

    PubMed Central

    Reithner, Barbara; Schuhmacher, Rainer; Stoppacher, Norbert; Pucher, Marion; Brunner, Kurt; Zeilinger, Susanne

    2015-01-01

    Trichoderma atroviride is a mycoparasite of a number of plant pathogenic fungi thereby employing morphological changes and secretion of cell wall degrading enzymes and antibiotics. The function of the tmk1 gene encoding a mitogen-activated protein kinase (MAPK) during fungal growth, mycoparasitic interaction, and biocontrol was examined in T. atroviride. Δtmk1 mutants exhibited altered radial growth and conidiation, and displayed de-regulated infection structure formation in the absence of a host-derived signal. In confrontation assays, tmk1 deletion caused reduced mycoparasitic activity although attachment to Rhizoctonia solani and Botrytis cinerea hyphae was comparable to the parental strain. Under chitinase-inducing conditions, nag1 and ech42 transcript levels and extracellular chitinase activities were elevated in a Δtmk1 mutant, whereas upon direct confrontation with R. solani or B. cinerea a host-specific regulation of ech42 transcription was found and nag1 gene transcription was no more inducible over an elevated basal level. Δtmk1 mutants exhibited higher antifungal activity caused by low molecular weight substances, which was reflected by an over-production of 6-pentyl-α-pyrone and peptaibol antibiotics. In biocontrol assays, a Δtmk1 mutant displayed a higher ability to protect bean plants against R. solani. PMID:17509915

  7. Evidence for a role of mitogen-activated protein kinases in the treatment of experimental acute pancreatitis

    PubMed Central

    Irrera, Natasha; Bitto, Alessandra; Interdonato, Monica; Squadrito, Francesco; Altavilla, Domenica

    2014-01-01

    Acute pancreatitis (AP) is an inflammatory disease characterized by acute inflammation and necrosis of the pancreatic parenchyma. AP is often associated with organ failure, sepsis, and high mortality. The pathogenesis of AP is still not well understood. In recent years several papers have highlighted the cellular and molecular events of acute pancreatitis. Pancreatitis is initiated by activation of digestive enzymes within the acinar cells that are involved in autodigestion of the gland, followed by a massive infiltration of neutrophils and macrophages and release of inflammatory mediators, responsible for the local and systemic inflammatory response. The hallmark of AP is parenchymal cell necrosis that represents the cause of the high morbidity and mortality, so that new potential therapeutic approaches are indispensable for the treatment of patients at high risk of complications. However, not all factors that determine the onset and course of the disease have been explained. Aim of this article is to review the role of mitogen-activated protein kinases in pathogenesis of acute pancreatitis. PMID:25469021

  8. The Roles of Mitogen-Activated Protein Kinase Pathways in TGF-β-Induced Epithelial-Mesenchymal Transition

    PubMed Central

    Gui, Ting; Sun, Yujing; Shimokado, Aiko; Muragaki, Yasuteru

    2012-01-01

    The mitogen-activated protein kinase (MAPK) pathway allows cells to interpret external signals and respond appropriately, especially during the epithelial-mesenchymal transition (EMT). EMT is an important process during embryonic development, fibrosis, and tumor progression in which epithelial cells acquire mesenchymal, fibroblast-like properties and show reduced intercellular adhesion and increased motility. TGF-β signaling is the first pathway to be described as an inducer of EMT, and its relationship with the Smad family is already well characterized. Studies of four members of the MAPK family in different biological systems have shown that the MAPK and TGF-β signaling pathways interact with each other and have a synergistic effect on the secretion of additional growth factors and cytokines that in turn promote EMT. In this paper, we present background on the regulation and function of MAPKs and their cascades, highlight the mechanisms of MAPK crosstalk with TGF-β signaling, and discuss the roles of MAPKs in EMT. PMID:22363839

  9. Signaling via the Trichoderma atroviride mitogen-activated protein kinase Tmk 1 differentially affects mycoparasitism and plant protection.

    PubMed

    Reithner, Barbara; Schuhmacher, Rainer; Stoppacher, Norbert; Pucher, Marion; Brunner, Kurt; Zeilinger, Susanne

    2007-11-01

    Trichoderma atroviride is a mycoparasite of a number of plant pathogenic fungi thereby employing morphological changes and secretion of cell wall degrading enzymes and antibiotics. The function of the tmk 1 gene encoding a mitogen-activated protein kinase (MAPK) during fungal growth, mycoparasitic interaction, and biocontrol was examined in T. atroviride. Deltatmk 1 mutants exhibited altered radial growth and conidiation, and displayed de-regulated infection structure formation in the absence of a host-derived signal. In confrontation assays, tmk 1 deletion caused reduced mycoparasitic activity although attachment to Rhizoctonia solani and Botrytis cinerea hyphae was comparable to the parental strain. Under chitinase-inducing conditions, nag 1 and ech 42 transcript levels and extracellular chitinase activities were elevated in a Deltatmk 1 mutant, whereas upon direct confrontation with R. solani or B. cinerea a host-specific regulation of ech 42 transcription was found and nag 1 gene transcription was no more inducible over an elevated basal level. Deltatmk 1 mutants exhibited higher antifungal activity caused by low molecular weight substances, which was reflected by an over-production of 6-pentyl-alpha-pyrone and peptaibol antibiotics. In biocontrol assays, a Deltatmk 1 mutant displayed a higher ability to protect bean plants against R. solani. PMID:17509915

  10. p38 mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes

    SciTech Connect

    Islam, Zahidul; Gray, Jennifer S.; Pestka, James J. . E-mail: pestka@msu.edu

    2006-06-15

    The effects of the ribotoxic trichothecene deoxynivalenol (DON) on mitogen-activated protein kinase (MAPK)-mediated IL-8 expression were investigated in cloned human monocytes and peripheral blood mononuclear cells (PBMC). DON (250 to 1000 ng/ml) induced both IL-8 mRNA and IL-8 heteronuclear RNA (hnRNA), an indicator of IL-8 transcription, in the human U937 monocytic cell line in a concentration-dependent manner. Expression of IL-8 hnRNA, mRNA and protein correlated with p38 phosphorylation and was completely abrogated by the p38 MAPK inhibitor SB203580. DON at 500 ng/ml similarly induced p38-dependent IL-8 protein and mRNA expression in PBMC cultures from healthy volunteers. Significantly increased IL-6 and IL-1{beta} intracellular protein and mRNA expression was also observed in PBMC treated with DON (500 ng/ml) which were also partially p38-dependent. Flow cytometry of PBMC revealed that DON-induced p38 phosphorylation varied among individuals relative to both threshold toxin concentrations (25-100 ng/ml) and relative increases in percentages of phospho-p38{sup +} cells. DON-induced p38 activation occurred exclusively in the CD14{sup +} monocyte population. DON was devoid of agonist activity for human Toll-like receptors 2, 3, 4, 5, 7, 8 and 9. However, two other ribotoxins, emetine and anisomycin, induced p38 phosphorylation in PBMC similarly to DON. Taken together, these data suggest that (1) p38 activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and (2) DON induced p38 activation in human monocytes via the ribotoxic stress response.

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

    PubMed

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

    2008-04-01

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

  12. Mitogen-activated protein kinase pathway mediates DBP-maf-induced apoptosis in RAW 264.7 macrophages.

    PubMed

    Gumireddy, Kiranmai; Reddy, C Damodar; Swamy, Narasimha

    2003-09-01

    Vitamin D-binding protein-macrophage-activating factor (DBP-maf) is derived from serum vitamin D binding protein (DBP) by selective deglycosylation during inflammation. In the present study, we investigated the effect of DBP-maf on RAW 264.7 macrophages and the underlying intracellular signal transduction pathways. DBP-maf increased proapoptotic caspase-3, -8, and -9 activities and induced apoptosis in RAW 264.7 cells. However, DBP, the precursor to DBP-maf did not induce apoptosis in these cells. Cell cycle analysis of DBP-maf-treated RAW 264.7 cells revealed growth arrest with accumulation of cells in sub-G(0)/G(1) phase. We also investigated the role of mitogen-activated protein kinase (MAPK) pathways in the DBP-maf-induced apoptosis of RAW264.7 cells. DBP-maf increased the phosphorylation of p38 and JNK1/2, while it decreased the ERK1/2 phosphorylation. Treatment with the p38 MAPK inhibitor, SB202190, attenuated DBP-maf-induced apoptosis. PD98059, a MEK specific inhibitor, did not show a significant inhibition of apoptosis induced by DBP-maf. Taken together, these results suggest that the p38 MAPK pathway plays a crucial role in DBP-maf-mediated apoptosis of macrophages. Our studies indicate that, during inflammation DBP-maf may function positively by causing death of the macrophages when activated macrophages are no longer needed at the site of inflammation. In summary, we report for the first time that DBP-maf induces apoptosis in macrophages via p38 and JNK1/2 pathway. PMID:12938159

  13. Neuropeptide Y1 receptor inhibits cell growth through inactivating mitogen-activated protein kinase signal pathway in human hepatocellular carcinoma.

    PubMed

    Lv, Xiufang; Zhao, Fengbo; Huo, Xisong; Tang, Weidong; Hu, Baoying; Gong, Xiu; Yang, Juan; Shen, Qiujin; Qin, Wenxin

    2016-07-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers, and its incidence is increasing worldwide. Neuropeptide Y (NPY) broadly expressed in the central and peripheral nervous system. It participates in multiple physiological and pathological processes through specific receptors. Evidences are accumulating that NPY is involved in development and progression in neuro- or endocrine-related cancers. However, little is known about the potential roles and underlying mechanisms of NPY receptors in HCC. In this study, we analyzed the expression of NPY receptors by real-time polymerase chain reaction, Western blot, and immunohistochemical staining. Correlation between NPY1R levels and clinicopathological characteristics, and survival of HCC patients were explored, respectively. Cell proliferation was researched by CCK-8 in vitro, and tumor growth was studied by nude mice xenografts in vivo. We found that mRNA and protein level of NPY receptor Y1 subtype (NPY1R) significantly decreased in HCC tissues. Low expression of NPY1R closely correlated with poor prognosis in HCC patients. Proliferation of HCC cells was significantly inhibited by recombinant NPY protein in vitro. This inhibitory effect could be blocked by selected NPY1R antagonist BIBP3226. Furthermore, overexpression of NPY1R could significantly inhibit HCC cell proliferation. Knockdown of NPY1R promoted cell multiplication in vitro and increased tumorigenicity and tumor growth in vivo. NPY1R was found to participate in the inhibition of cell proliferation via inactivating mitogen-activated protein kinase signal pathway in HCC cells. Collectively, NPY1R plays an inhibitory role in tumor growth and may be a promising therapeutic target for HCC. PMID:27262566

  14. Nodal promotes invasive phenotypes via a Mitogen Activated Protein Kinase-dependent pathway

    PubMed Central

    Quail, DF; Zhang, G; Findlay, SD; Hess, DA; Postovit, LM

    2016-01-01

    The progression of cancer from localized to invasive disease is requisite for metastasis, and is often characterized by epithelial-to-mesenchymal transition (EMT) and alterations in cellular adhesion and migration. Studies have shown that this transition is associated with an up-regulation of embryonic stem cell-associated genes, resulting in a dedifferentiated phenotype and poor patient prognosis. Nodal is an embryonic factor that plays a critical role in promoting early invasive events during development. Nodal is silenced as stem cells differentiate; however, it re-emerges in adult life during placentation and mammary gland development, and is aberrantly expressed in many cancers. Here, we show that Nodal over-expression, in poorly-invasive breast cancer and choriocarcinoma cells, causes increased invasion and migration in vitro. Furthermore, we show that Nodal over-expression in these epithelial cancer types induces an EMT-like event concomitant with the internalization of E-Cadherin. This ability of Nodal to promote cellular invasion and EMT-like phenomena is dependent upon the phosphorylation of ERK1/2. Since Nodal normally signals through SMADs, these findings lend insight into an alternative pathway that is hijacked by this protein in cancer. To evaluate the clinical implications of our results, we show that Nodal inhibition reduces liver tumor burden in a model of spontaneous breast cancer metastasis in vivo, and that Nodal loss-of-function in aggressive breast cancer lines results in a decrease in invasive phenotypes. Our results demonstrate that Nodal is involved in promoting invasion in multiple cellular contexts, and that Nodal inhibition may be useful as a therapeutic target for patients with progressive disease. PMID:23334323

  15. Role of mitogen activated protein kinases in skin tumorigenicity of Patulin

    SciTech Connect

    Saxena, Neha; Ansari, Kausar M.; Kumar, Rahul; Chaudhari, Bhushan P.; Dwivedi, Premendra D.; Das, Mukul

    2011-12-15

    WHO has highlighted the need to evaluate dermal toxicity of mycotoxins including Patulin (PAT), detected in several fruits. In this study the skin carcinogenic potential of topically applied PAT was investigated. Single topical application of PAT (400 nmol) showed enhanced cell proliferation ({approx} 2 fold), along with increased generation of ROS and activation of ERK, p38 and JNK MAPKs, in mouse skin. PAT exposure also showed activation of downstream target proteins, c-fos, c-Jun and NF-{kappa}B transcription factors. Further, single topical application of PAT (400 nmol) followed by twice weekly application of TPA resulted in tumor formation after 14 weeks, indicating the tumor initiating activity of PAT. However no tumors were observed when PAT was used either as a complete carcinogen (80 nmol) or as a tumor promoter (20 nmol and 40 nmol) for 25 weeks. Histopathological findings of tumors found in PAT/TPA treated mice showed that these tumors were of squamous cell carcinoma type and similar to those found in the positive control group (DMBA/TPA) along with significant increase of lipid peroxidation and decrease in free sulfydryls, catalase, superoxide dismutase and glutathione reductase activities. The results suggest the possible role of free radicals in PAT mediated dermal tumorigenicity involving MAPKs. -- Highlights: Black-Right-Pointing-Pointer Single topical application of Patulin showed enhanced cell proliferation. Black-Right-Pointing-Pointer Patulin activate MAPKs, c-fos, c-Jun and NF-{kappa}B transcription factors. Black-Right-Pointing-Pointer Patulin showed skin tumor initiating potential. Black-Right-Pointing-Pointer We could not detect skin tumor promoting potential of Patulin at the tested dose. Black-Right-Pointing-Pointer However prolonged exposure of Patulin at a higher dose may promote tumor.

  16. Activation of ERK mitogen-activated protein kinase in human cells by the mycotoxin patulin

    SciTech Connect

    Wu, T.-S.; Yu, F.-Y.; Su, C.-C.; Kan, J.-C.; Chung, C.-P.; Liu, B.-H. . E-mail: bingliu@csmu.edu.tw

    2005-09-01

    Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration-dependent and time-dependent increase in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human embryonic kidney (HEK293) cells, human peripheral blood mononuclear cells (PBMCs), and Madin-Darby canine kidney (MDCK) cells. Exposure of HEK293 cells to concentrations above 5 {mu}M PAT for 30 min induced ERK1/2 phosphorylation; activation of ERK1/2 was also observed after 24 h incubation with 0.05 {mu}M of PAT. Treatment of human PBMCs for 30 min with 30 {mu}M PAT dramatically increased the phosphorylated ERK1/2 levels. Both MEK1/2 inhibitors, U0126 and PD98059, suppressed ERK1/2 activation in either HEK293 or MDCK cells. In HEK293 cells, U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage, expressed as tail moment values, in the single cell gel electrophoresis assay. Conversely, U0126 did not affect cell viability, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated cultures. Exposure of HEK293 cells for 90 min to 15 {mu}M PAT elevated the levels of early growth response gene-1 (egr-1) mRNA, but not of c-fos, fosB, and junB mRNAs. These results indicate that in human cells, PAT causes a rapid and persistent activation of ERK1/2 and this signaling pathway plays an important role in mediating PAT-induced DNA damage and egr-1 gene expression.

  17. Surfactant protein D induces immune quiescence and apoptosis of mitogen-activated peripheral blood mononuclear cells.

    PubMed

    Pandit, Hrishikesh; Thakur, Gargi; Koippallil Gopalakrishnan, Aghila Rani; Dodagatta-Marri, Eswari; Patil, Anushree; Kishore, Uday; Madan, Taruna

    2016-02-01

    Surfactant protein D (SP-D) is an integral molecule of the innate immunity secreted by epithelial cells lining the mucosal surfaces. The C-type lectin domain of SP-D performs pattern recognition functions while it binds to putative receptors on immune cells to modify cellular functions. Activation of immune cells and increased serum SP-D is observed in a range of patho-physiological conditions including infections. We speculated if SP-D can modulate systemic immune response via direct interaction with activated PBMCs. In this study, we examined interaction of a recombinant fragment of human SP-D (rhSP-D) on PHA-activated PBMCs. We report a significant downregulation of activation receptors such as TLR2, TLR4, CD11c and CD69 upon rhSP-D treatment. rhSP-D inhibited production of Th1 (TNF-α and IFN-γ) and Th17 (IL-17A) cytokines along with IL-6. Interestingly, levels of IL-2, Th2 (IL-4) and regulatory (IL-10 and TGF-β) cytokines remained unaltered. Analysis of co-stimulatory CD28 and co-inhibitory CTLA4 receptors along with their ligands CD80 and CD86 revealed a selective up-regulation of CTLA4 in the lymphocyte subset. rhSP-D induced apoptosis in the activated but not in non-activated lymphocytes. Blockade of CTLA4 inhibited rhSP-D mediated apoptosis of activated lymphocytes, confirming involvement of CTLA4. We conclude that SP-D restores immune homeostasis. It regulates expression of immunomodulatory receptors and cytokines, which is followed by induction of apoptosis in activated lymphocytes. These findings suggest a critical role of SP-D in immune surveillance against activated immune cells. PMID:26563748

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

    PubMed

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

    2001-06-01

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

  19. Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

    PubMed Central

    Juo, P; Kuo, C J; Reynolds, S E; Konz, R F; Raingeaud, J; Davis, R J; Biemann, H P; Blenis, J

    1997-01-01

    The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades. PMID:8972182

  20. Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi.

    PubMed

    Lew, Roger R; Levina, Natalia N; Shabala, Lana; Anderca, Marinela I; Shabala, Sergey N

    2006-03-01

    Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca(2+) influx and the sustained hyperpolarization is due to H(+) efflux by activation of the plasma membrane H(+)-ATPase. Protein synthesis is not required for H(+)-ATPase activation. Net K(+) and Cl(-) uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl(-) uptake increases, but net K(+) flux barely changes and net H(+) efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H(+)-ATPase, and net K(+) and Cl(-) uptake during turgor regulation. Other pathways regulating turgor must also exist. PMID:16524903

  1. Role of a Mitogen-Activated Protein Kinase Cascade in Ion Flux-Mediated Turgor Regulation in Fungi

    PubMed Central

    Lew, Roger R.; Levina, Natalia N.; Shabala, Lana; Anderca, Marinela I.; Shabala, Sergey N.

    2006-01-01

    Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca2+ influx and the sustained hyperpolarization is due to H+ efflux by activation of the plasma membrane H+-ATPase. Protein synthesis is not required for H+-ATPase activation. Net K+ and Cl− uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl− uptake increases, but net K+ flux barely changes and net H+ efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H+-ATPase, and net K+ and Cl− uptake during turgor regulation. Other pathways regulating turgor must also exist. PMID:16524903

  2. Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells.

    PubMed Central

    Malarkey, K.; Chilvers, E. R.; Lawson, M. F.; Plevin, R.

    1995-01-01

    1. In cultures of bovine tracheal smooth muscle cells, platelet-derived growth factor-BB (PDGF), bradykinin (BK) and endothelin-1 (ET-1) stimulated the tyrosine phosphorylation and activation of both pp42 and pp44 kDa forms of mitogen-activated protein (MAP) kinase. 2. Both ET-1 and PDGF stimulated a sustained activation of MAP kinase whilst the response to BK was transient. 3. Activation of MAP kinase occurred in a concentration-dependent manner (EC50 values: ET-1, 2.3 +/- 1.3 nM; BK, 8.7 +/- 4.1 nM, PDGF, 9.7 +/- 3.2 ng ml-1). 4. Pretreatment with the protein kinase C (PKC) inhibitor Ro-318220, significantly reduced ET-1 activation of MAP kinase at 2 and 5 min but enhanced MAP kinase activation at 60 min. 5. Following chronic phorbol ester pretreatment, BK-stimulated activation of MAP kinase was abolished whilst the responses to PDGF and ET-1 were only partly reduced (80 and 45% inhibition respectively). 6. Pretreatment with pertussis toxin reduced ET-1 stimulated activation of MAP kinase particularly at later times (60 min), but left the responses to both PDGF and BK unaffected. 7. ET-1 also stimulated a 3 fold increase in [3H]-thymidine incorporation which was abolished by pertussis toxin pretreatment. In contrast, PDGF stimulated a 131 fold increase in [3H]-thymidine incorporation which was not affected by pertussis toxin. 8. These results suggest that a pertussis toxin-sensitive activation of MAP kinase may play an important role in ET-1-stimulated DNA synthesis but that activation of MAP kinase alone is not sufficient to induce the magnitude of DNA synthesis observed in response to PDGF. Images Figure 1 Figure 2 Figure 5 Figure 6 Figure 7 PMID:8564258

  3. Expression of mitogen-activated protein kinase phosphatase-1 in the early phases of human epithelial carcinogenesis.

    PubMed Central

    Loda, M.; Capodieci, P.; Mishra, R.; Yao, H.; Corless, C.; Grigioni, W.; Wang, Y.; Magi-Galluzzi, C.; Stork, P. J.

    1996-01-01

    Many mitogens and human oncogenes activate extracellular regulated kinases (ERKs), which in turn convey proliferation signals. ERKs or mitogen-activated protein (MAP) kinases are inactivated in vitro by MAP kinase phosphatases (MKPs). The gene encoding one of these MKPs, MKP-1, is a serum-inducible gene and is transcriptionally activated by mitogenic signals in cultured cells. As MKP-1 has been shown to block DNA synthesis by inhibiting ERKs when expressed at elevated levels in cultured cells, it has been suggested that it may act as a tumor suppressor. MKP-1 mRNA and MAP kinase (ERK-1 and -2) protein expression was assessed in 164 human epithelial tumors of diverse tissue origin by in situ hybridization and immunohistochemistry. MKP-1 was overexpressed in the early phases of prostate, colon, and bladder carcinogenesis, with progressive loss of expression with higher histological grade and in metastases. In contrast, breast carcinomas showed significant MKP-1 expression even when poorly differentiated or in late stages of the disease. MKP-1, ERK-1, and ERK-2 were co-expressed in most tumors examined. In a subset of 15 tumors, ERK-1 enzymatic activity as well as structural alterations that might be responsible for loss of function of MKP-1 during tumor progression, were examined. ERK-1 enzymatic activity was found to be elevated despite MKP-1 overexpression. No loss of 5q35-ter (containing the MKP-1 locus) was detected by polymerase chain reaction in metastases compared with primary tumors. Finally, no mutations were found in the catalytic domain of MKP-1. These data indicate that MKP-1 is an early marker for a wide range of human epithelial tumors and suggest that MKP-1 does not behave as a tumor suppressor in epithelial tumors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8909245

  4. KIF5B transports BNIP-2 to regulate p38 mitogen-activated protein kinase activation and myoblast differentiation

    PubMed Central

    Yi, Peng; Chew, Li Li; Zhang, Ziwang; Ren, Hao; Wang, Feiya; Cong, Xiaoxia; Zheng, Liling; Luo, Yan; Ouyang, Hongwei; Low, Boon Chuan; Zhou, Yi Ting

    2015-01-01

    The Cdo-p38MAPK (p38 mitogen-activated protein kinase) signaling pathway plays important roles in regulating skeletal myogenesis. During myogenic differentiation, the cell surface receptor Cdo bridges scaffold proteins BNIP-2 and JLP and activates p38MAPK, but the spatial-temporal regulation of this process is largely unknown. We here report that KIF5B, the heavy chain of kinesin-1 motor, is a novel interacting partner of BNIP-2. Coimmunoprecipitation and far-Western study revealed that BNIP-2 directly interacted with the motor and tail domains of KIF5B via its BCH domain. By using a range of organelle markers and live microscopy, we determined the endosomal localization of BNIP-2 and revealed the microtubule-dependent anterograde transport of BNIP-2 in C2C12 cells. The anterograde transport of BNIP-2 was disrupted by a dominant-negative mutant of KIF5B. In addition, knockdown of KIF5B causes aberrant aggregation of BNIP-2, confirming that KIF5B is critical for the anterograde transport of BNIP-2 in cells. Gain- and loss-of-function experiments further showed that KIF5B modulates p38MAPK activity and in turn promotes myogenic differentiation. Of importance, the KIF5B-dependent anterograde transport of BNIP-2 is critical for its promyogenic effects. Our data reveal a novel role of KIF5B in the spatial regulation of Cdo–BNIP-2–p38MAPK signaling and disclose a previously unappreciated linkage between the intracellular transporting system and myogenesis regulation. PMID:25378581

  5. The transcription factor FOXF1 promotes prostate cancer by stimulating the mitogen-activated protein kinase ERK5.

    PubMed

    Fulford, Logan; Milewski, David; Ustiyan, Vladimir; Ravishankar, Navin; Cai, Yuqi; Le, Tien; Masineni, Sreeharsha; Kasper, Susan; Aronow, Bruce; Kalinichenko, Vladimir V; Kalin, Tanya V

    2016-01-01

    Forkhead box F1 (FOXF1) is a stromal transcription factor that is not expressed in epithelial cells of normal prostate tissue. The role of FOXF1 in cancer is conflicting; its loss in some cancers suggests a tumor suppressive function, but its abundance in others is associated with protumorigenic and metastatic traits. Extracellular signal-regulated kinase 5 (ERK5) is associated with advanced-stage prostate adenocarcinoma (PCa) in patients. We detected a population of FOXF1-positive tumor cells in aggressive mouse and human PCa. Using two murine orthotopic models of PCa, we found that overexpression of FOXF1 in Myc-CaP and TRAMP prostate tumor cells induced tumor growth in the prostate and progression to peritoneal metastasis. Increased growth of FOXF1-positive prostate tumors was associated with increased phosphorylation of ERK5, a member of the mitogen-activated protein kinase (MAPK) family. FOXF1 transcriptionally induced and directly bound to promoter regions of genes encoding the kinases MAP3K2 and WNK1, which promoted the phosphorylation and activation of ERK5. Knockdown of ERK5 or both MAP3K2 and WNK1 in FOXF1-overexpressing PCa cells reduced cell proliferation in culture and suppressed tumor growth and tumor metastasis when implanted into mice. In human tumors, FOXF1 expression correlated positively with that of MAP3K2 and WNK1 Thus, in contrast to some tumors where FOXF1 may function as a tumor suppressor, FOXF1 promotes prostate tumor growth and progression by activating ERK5 signaling. Our results also indicate that ERK5 may be a new therapeutic target in patients with FOXF1-positive PCa. PMID:27165781

  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. Lipopolysaccharide induces cholangiocyte proliferation via an interleukin-6-mediated activation of p44/p42 mitogen-activated protein kinase.

    PubMed

    Park, J; Gores, G J; Patel, T

    1999-04-01

    The biliary epithelium is exposed to mediators of inflammation such as bacterial endotoxin or lipopolysaccharide (LPS) in a variety of inflammatory conditions. These conditions are also characterized by cholangiocyte proliferation and a predisposition to malignancy. Furthermore, LPS can enhance the expression of interleukin-6 (IL-6), a known biliary mitogen. However, the effects of LPS on cholangiocyte proliferation or IL-6 secretion are unknown. Thus, our aims were to determine if LPS stimulates cholangiocyte proliferation by IL-6-dependent signaling pathways. H69 cells derived from normal human intrahepatic cholangiocytes proliferated in response to LPS. Cholangiocytes responded to LPS (and other inflammatory cytokines such as tumor necrosis factor alpha [TNF-alpha] and IL-1beta) by increased secretion of IL-6, which had a mitogenic effect on H69 cells. Preincubation with anti-IL-6 neutralizing antibodies inhibited LPS-induced proliferation. Furthermore, cholangiocytes possessed the IL-6 receptor complex subunits and intact signaling mechanisms leading to activation of signal transducers and activators of transcription (STAT) factors. Although both p38 and p44/p42 mitogen-activated protein kinases (MAPKs) were constitutively present and active in cholangiocytes, IL-6 increased p44/p42, but not p38 MAPK activity. PD098059 inhibited activation of p44/p42 MAPK in cholangiocytes and completely blocked DNA synthesis in response to IL-6 or LPS. These studies identify a critical role for the p44/p42 MAPK in cholangiocyte proliferation and demonstrate that the proliferative response of cholangiocytes to inflammatory mediators such as LPS involves IL-6-mediated activation of the p44/p42 MAPK pathway. PMID:10094943

  8. Vitexin protects brain against ischemia/reperfusion injury via modulating mitogen-activated protein kinase and apoptosis signaling in mice.

    PubMed

    Wang, Yanan; Zhen, Yilan; Wu, Xian; Jiang, Qin; Li, Xiaoliang; Chen, Zhiwu; Zhang, Gongliang; Dong, Liuyi

    2015-03-15

    Vitexin is a major bioactive flavonoid compound derived from the dried leaf of hawthorn (Crataegus pinnatifida), a widely used conventional folk medicine in China. Recent studies have shown that vitexin presents neuroprotective effects in vitro. Whether this protective effect applies to the cerebral ischemia/reperfusion (I/R) injury remains elusive. In the present study, we examined the potential neuroprotective effect of vitexin against cerebral I/R injury and underlying mechanisms. A focal cerebral I/R model in male Kunming mice was induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 22 h. The neurological function and infarct volume were assessed by using Long's five-point scale system and triphenyl-tetrazolium chloride (TTC) staining technique, respectively. Neuronal damage was evaluated by histological staining. Extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 phosphorylation, and apoptosis were measured via Western blot at 24 h after reperfusion. As a result, systemic vitexin treatment significantly reduced neurological deficit, cerebral infarct volume and neuronal damage when compared with the I/R group. Western blot analyses revealed that vitexin markedly upregulated p-ERK1/2 and downregulated p-JNK and p-p38. Meanwhile, vitexin increased Bcl-2 expression and suppressed the overexpression of Bax in the I/R injury mice. In conclusion, the results indicate that vitexin protects brain against cerebral I/R injury, and this effect may be regulated by mitogen-activated protein kinase (MAPK) and apoptosis signaling pathways. PMID:25837275

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

    PubMed Central

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

    2011-01-01

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

  10. Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.)

    PubMed Central

    Sun, Yun; Wang, Chen; Yang, Bo; Jiang, Yuan-Qing

    2014-01-01

    Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A–C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription–PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola. PMID:24604738

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

    PubMed

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

    2015-11-01

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

  12. Local anesthetics induce apoptosis in human thyroid cancer cells through the mitogen-activated protein kinase pathway.

    PubMed

    Chang, Yuan-Ching; Hsu, Yi-Chiung; Liu, Chien-Liang; Huang, Shih-Yuan; Hu, Meng-Chun; Cheng, Shih-Ping

    2014-01-01

    Local anesthetics are frequently used in fine-needle aspiration of thyroid lesions and locoregional control of persistent or recurrent thyroid cancer. Recent evidence suggests that local anesthetics have a broad spectrum of effects including inhibition of cell proliferation and induction of apoptosis in neuronal and other types of cells. In this study, we demonstrated that treatment with lidocaine and bupivacaine resulted in decreased cell viability and colony formation of both 8505C and K1 cells in a dose-dependent manner. Lidocaine and bupivacaine induced apoptosis, and necrosis in high concentrations, as determined by flow cytometry. Lidocaine and bupivacaine caused disruption of mitochondrial membrane potential and release of cytochrome c, accompanied by activation of caspase 3 and 7, PARP cleavage, and induction of a higher ratio of Bax/Bcl-2. Based on microarray and pathway analysis, apoptosis is the prominent transcriptional change common to lidocaine and bupivacaine treatment. Furthermore, lidocaine and bupivacaine attenuated extracellular signal-regulated kinase 1/2 (ERK1/2) activity and induced activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase. Pharmacological inhibitors of MAPK/ERK kinase and p38 MAPK suppressed caspase 3 activation and PARP cleavage. Taken together, our results for the first time demonstrate the cytotoxic effects of local anesthetics on thyroid cancer cells and implicate the MAPK pathways as an important mechanism. Our findings have potential clinical relevance in that the use of local anesthetics may confer previously unrecognized benefits in the management of patients with thyroid cancer. PMID:24586874

  13. Roles of mitogen activated protein kinases and EGF receptor in arsenite-stimulated matrix metalloproteinase-9 production

    SciTech Connect

    Cooper, Karen L.; Myers, Terrance Alix; Rosenberg, Martina; Chavez, Miquella; Hudson, Laurie G. . E-mail: lghudson@unm.edu

    2004-11-01

    The dermatotoxicity of arsenic is well established and epidemiological studies identify an increased incidence of keratinocytic tumors (basal cell and squamous cell carcinoma) associated with arsenic exposure. Little is known about the underlying mechanisms of arsenic-mediated skin carcinogenesis, but activation of mitogen-activated protein (MAP) kinases and subsequent regulation of downstream target genes may contribute to tumor promotion and progression. In this study, we investigated activation of the extracellular signal regulated kinase (ERK) and the stress-associated kinase p38 by arsenite in HaCat cells, a spontaneously immortalized human keratinocyte cell line. Arsenite concentrations {>=}100 {mu}M stimulate rapid activation of p38 and ERK MAP kinases. However, upon extended exposure (24 h), persistent stimulation of p38 and ERK MAP kinases was detected at low micromolar concentrations of arsenite. Although ERK and p38 were activated with similar time and concentration dependence, the mechanism of activation differed for these two MAP kinases. ERK activation by arsenite was fully dependent on the catalytic activity of the epidermal growth factor (EGF) receptor and partially dependent on Src-family kinase activity. In contrast, p38 activation was independent of EGF receptor or Src-family kinase activity. Arsenite-stimulated MAP kinase signal transduction resulted in increased production of matrix metalloproteinase (MMP)-9, an AP-1 regulated gene product. MMP-9 induction by arsenite was prevented when EGF receptor or MAP kinase signaling was inhibited. These studies indicate that EGF receptor activation is a component of arsenite-mediated signal transduction and gene expression in keratinocytes and that low micromolar concentrations of arsenite stimulate key signaling pathways upon extended exposure. Stimulation of MAP kinase cascades by arsenic and subsequent regulation of genes including c-fos, c-jun, and the matrix degrading proteases may play an important

  14. Contributions of reactive oxygen species and mitogen-activated protein kinase signaling in arsenite-stimulated hemeoxygenase-1 production

    SciTech Connect

    Cooper, Karen L.; Liu, Ke Jian; Hudson, Laurie G. . E-mail: lhudson@salud.unm.edu

    2007-01-15

    Hemeoxygenase-1 (HO-1) is an oxidative stress responsive gene upregulated by various physiological and exogenous stimuli. HO-1 has cytoprotective activities and arsenite is a potent inducer of HO-1 in many cell types and tissues, including epidermal keratinocytes. We investigated the potential contributions of reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) activation to arsenite-dependent regulation of HO-1 in HaCaT cells, an immortalized human keratinocyte line. Both epidermal growth factor (EGF) and arsenite stimulated ROS production was detected by dihydroethidium (DHE) staining and fluorescence microscopy. Arsenite induced HO-1 in a time- and concentration-dependent manner, while HO-1 expression in response to EGF was modest and evident at extended time points (48-72 h). Inhibition of EGF receptor, MEK I/II or Src decreased arsenite-stimulated HO-1 expression by 20-30%. In contrast, addition of a superoxide scavenger or inhibition of p38 activity decreased the arsenite-dependent response by 80-90% suggesting that ROS and p38 are required for HO-1 induction. However, ROS generation alone was insufficient for the observed arsenite-dependent response as use of a xanthine/xanthine oxidase system to generate ROS did not produce an equivalent upregulation of HO-1. Cooperation between ERK signaling and ROS generation was demonstrated by synergistic induction of HO-1 in cells co-treated with EGF and xanthine/xanthine oxidase resulting in a response nearly equivalent to that observed with arsenite. These findings suggest that the ERK/MAPK activation is necessary but not sufficient for optimal arsenite-stimulated HO-1 induction. The robust and persistent upregulation of HO-1 may have a role in cellular adaptation to chronic arsenic exposure.

  15. Mitogen activated protein kinases SakA(HOG1) and MpkC collaborate for Aspergillus fumigatus virulence.

    PubMed

    Bruder Nascimento, Ariane Cristina Mendes de Oliveira; Dos Reis, Thaila Fernanda; de Castro, Patrícia Alves; Hori, Juliana I; Bom, Vinícius Leite Pedro; de Assis, Leandro José; Ramalho, Leandra Naira Zambelli; Rocha, Marina Campos; Malavazi, Iran; Brown, Neil Andrew; Valiante, Vito; Brakhage, Axel A; Hagiwara, Daisuke; Goldman, Gustavo H

    2016-06-01

    Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen-activated protein kinases of the high-osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild-type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis. PMID:26878695

  16. Curcumin increases gelatinase activity in human neutrophils by a p38 mitogen-activated protein kinase (MAPK)-independent mechanism.

    PubMed

    Antoine, Francis; Girard, Denis

    2015-01-01

    Curcumin has been found to possess anti-inflammatory activities and neutrophils, key players in inflammation, were previously found to be important targets to curcumin in a few studies. For example, curcumin was found to induce apoptosis in neutrophils by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. However, the role of curcumin on the biology of neutrophils is still poorly defined. To study the role of curcumin on neutrophil degranulation and to determine the role of p38 MAPK, human neutrophils were freshly isolated from healthy individuals and incubated in vitro with curcumin. Degranulation was studied at three levels: surface expression of granule markers by flow cytometry; release of matrix metallopeptidase-9 (MMP-9 or gelatinase B) enzyme into supernatants by Western blot; and gelatinase B activity by zymography. Activation of p38 MAPK was studied by monitoring its tyrosine phosphorylation levels by western blot and its role by the utilization of a pharmacological inhibitor. The results indicate that curcumin increased the cell surface expression of CD35 (secretory vesicle), CD63 (azurophilic granules), and CD66b (gelatinase granules) in neutrophils. Also, curcumin increased the release and enzymatic activity of gelatinase B in the extracellular milieu and activated p38 MAP kinase in these cells. However, in contrast to fMLP, curcumin-induced enzymatic activity and secretion of gelatinase B were not reversed by use of a p38 inhibitor. Finally, it was found that curcumin was able to enhance phagocytosis. Taken together, the results here demonstrate that curcumin induced degranulation in human neutrophils and that the increased gelatinase activity is not dependent on p38 MAPK activation. Therefore, degranulation is another human neutrophil function that could be modulated by curcumin, as well as phagocytosis. PMID:24926560

  17. Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).

    PubMed

    Sun, Yun; Wang, Chen; Yang, Bo; Wu, Feifei; Hao, Xueyu; Liang, Wanwan; Niu, Fangfang; Yan, Jingli; Zhang, Hanfeng; Wang, Boya; Deyholos, Michael K; Jiang, Yuan-Qing

    2014-05-01

    Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola. PMID:24604738

  18. Activation of transcription factor AP-1 and mitogen-activated protein kinases in aniline-induced splenic toxicity

    SciTech Connect

    Khan, M. Firoze . E-mail: mfkhan@utmb.edu; Kannan, Subburaj; Wang Jianling

    2006-01-15

    Signaling mechanisms in aniline-induced fibrogenic and/or tumorigenic response in the spleen are not known. Previous studies have shown that aniline exposure leads to iron accumulation and oxidative stress in the spleen, which may cause activation of redox-sensitive transcription factors and regulate the transcription of genes involved in fibrosis and/or tumorigenesis. To test this, male SD rats were treated with 0.5 mmol/kg/day aniline via drinking water for 30 days, and activation of transcription factor AP-1 was determined in the splenocyte nuclear extracts (NEs). AP-1 DNA-binding activity in the NEs of freshly isolated splenocytes from aniline-treated rats increased in comparison to the controls, as determined by electrophoretic mobility shift assay (EMSA). AP-1 binding was also determined in the NEs of cultured splenocytes (2 h and 24 h), which showed even a greater increase in binding activity at 2 h. The specificity of AP-1 binding for relevant DNA motifs was confirmed by competition EMSA and by supershift EMSA using antibodies specific to c-Jun and c-Fos. To further explore the signaling mechanisms in the AP-1 activation, phosphorylation patterns of mitogen-activated protein kinases (MAPKs) were pursued. Aniline exposure induced increases in the phosphorylation of the three classes of MAPKs: extracellular-signal-regulated kinase (ERK 1/2), c-Jun N-terminal kinase (JNK 1/2), and p38 MAPKs. Furthermore, TGF-{beta}1 mRNA expression showed a 3-fold increase in the spleens of aniline-treated rats. These observations suggest a strong association among MAPK phosphorylation, AP-1 activation, and enhanced TGF-{beta}1 gene expression. The observed sequence of events subsequent to aniline exposure could regulate genes that lead to fibrogenic and/or tumorigenic response in the spleen.

  19. Upstream mitogen-activated protein kinase (MAPK) pathway inhibition: MEK inhibitor followed by a BRAF inhibitor in advanced melanoma patients.

    PubMed

    Goldinger, Simone M; Zimmer, Lisa; Schulz, Carsten; Ugurel, Selma; Hoeller, Christoph; Kaehler, Katharina C; Schadendorf, Dirk; Hassel, Jessica C; Becker, Juergen; Hauschild, Axel; Dummer, Reinhard

    2014-01-01

    BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients. BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi. A total of 23 melanoma patients (six females, 17 males, aged 47-80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2-23.2) and subsequent for BRAFi 4.5 (1.2-15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed. Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies. PMID:24183461

  20. Upregulation of early growth response factor-1 by bile acids requires mitogen-activated protein kinase signaling

    SciTech Connect

    Allen, Katryn; Kim, Nam Deuk; Moon, Jeon-OK; Copple, Bryan L.

    2010-02-15

    Cholestasis results when excretion of bile acids from the liver is interrupted. Liver injury occurs during cholestasis, and recent studies showed that inflammation is required for injury. Our previous studies demonstrated that early growth response factor-1 (Egr-1) is required for development of inflammation in liver during cholestasis, and that bile acids upregulate Egr-1 in hepatocytes. What remains unclear is the mechanism by which bile acids upregulate Egr-1. Bile acids modulate gene expression in hepatocytes by activating the farnesoid X receptor (FXR) and through activation of mitogen-activated protein kinase (MAPK) signaling. Accordingly, the hypothesis was tested that bile acids upregulate Egr-1 in hepatocytes by FXR and/or MAPK-dependent mechanisms. Deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) stimulated upregulation of Egr-1 to the same extent in hepatocytes isolated from wild-type mice and FXR knockout mice. Similarly, upregulation of Egr-1 in the livers of bile duct-ligated (BDL) wild-type and FXR knockout mice was not different. Upregulation of Egr-1 in hepatocytes by DCA and CDCA was prevented by the MEK inhibitors U0126 and SL-327. Furthermore, pretreatment of mice with U0126 prevented upregulation of Egr-1 in the liver after BDL. Results from these studies demonstrate that activation of MAPK signaling is required for upregulation of Egr-1 by bile acids in hepatocytes and for upregulation of Egr-1 in the liver during cholestasis. These studies suggest that inhibition of MAPK signaling may be a novel therapy to prevent upregulation of Egr-1 in liver during cholestasis.

  1. Mitogen-activated protein kinase kinases promote mitochondrial biogenesis in part through inducing peroxisome proliferator-activated receptor γ coactivator-1β expression.

    PubMed

    Gao, Minghui; Wang, Junjian; Lu, Na; Fang, Fang; Liu, Jinsong; Wong, Chi-Wai

    2011-06-01

    Growth factor activates mitogen-activated protein kinase kinases to promote cell growth. Mitochondrial biogenesis is an integral part of cell growth. How growth factor regulates mitochondrial biogenesis is not fully understood. In this study, we found that mitochondrial mass was specifically reduced upon serum starvation and induced upon re-feeding with serum. Using mitogen-activated protein kinase kinases inhibitor U0126, we found that the mRNA expression levels of ATP synthase, cytochrome-C, mitochondrial transcription factor A, and mitofusin 2 were reduced. Since the transcriptional levels of these genes are under the control of peroxisome proliferator-activated receptor γ coactivator-1α and -1β (PGC-1α and PGC-1β), we examined and found that only the mRNA and protein levels of PGC-1β were suppressed. Importantly, over-expression of PGC-1β partially reversed the reduction of mitochondrial mass upon U0126 treatment. Thus, we conclude that mitogen-activated protein kinase kinases direct mitochondrial biogenesis through selectively inducing PGC-1β expression. PMID:21458501

  2. Cell-Associated Hemolysis Induced by Helicobacter pylori Is Mediated by Phospholipases with Mitogen-Activated Protein Kinase-Activating Properties

    PubMed Central

    Sitaraman, Ramakrishnan; Israel, Dawn A.; Romero-Gallo, Judith

    2012-01-01

    Pathogenic Helicobacter pylori strains can selectively activate epithelial mitogen-activated protein kinase (MAPK) signaling pathways linked with disease. We now demonstrate that H. pylori-induced hemolysis is strain specific and is mediated by phospholipases PldA1 and PldD. Inactivation of PldD inhibited activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), indicating that H. pylori hemolytic phospholipases also harbor MAPK-activating properties. PMID:22205825

  3. Cell-associated hemolysis induced by Helicobacter pylori is mediated by phospholipases with mitogen-activated protein kinase-activating properties.

    PubMed

    Sitaraman, Ramakrishnan; Israel, Dawn A; Romero-Gallo, Judith; Peek, Richard M

    2012-03-01

    Pathogenic Helicobacter pylori strains can selectively activate epithelial mitogen-activated protein kinase (MAPK) signaling pathways linked with disease. We now demonstrate that H. pylori-induced hemolysis is strain specific and is mediated by phospholipases PldA1 and PldD. Inactivation of PldD inhibited activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), indicating that H. pylori hemolytic phospholipases also harbor MAPK-activating properties. PMID:22205825

  4. Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

    2014-01-01

    The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

  5. Mitogen-activated protein kinases are associated with the regulation of physiological traits and virulence in Fusarium oxysporum f. sp. cubense.

    PubMed

    Ding, Zhaojian; Li, Minhui; Sun, Fei; Xi, Pinggen; Sun, Longhua; Zhang, Lianhui; Jiang, Zide

    2015-01-01

    Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that null mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence. PMID:25849862

  6. Mitogen-Activated Protein Kinases Are Associated with the Regulation of Physiological Traits and Virulence in Fusarium oxysporum f. sp. cubense

    PubMed Central

    Ding, Zhaojian; Li, Minhui; Sun, Fei; Xi, Pinggen; Sun, Longhua; Zhang, Lianhui; Jiang, Zide

    2015-01-01

    Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that null mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence. PMID:25849862

  7. Mutations associated with retinopathies alter mitogen-activated protein kinase-induced phosphorylation of neural retina leucine-zipper

    PubMed Central

    Kumar, Sandeep; Patel, Dharmesh; Richong, Sushmita; Oberoi, Pranav; Ghosh, Madhumita; Swaroop, Anand

    2007-01-01

    Purpose Neural retina leucine-zipper (NRL), a member of the basic motif leucine zipper family of transcription factors, is preferentially expressed in rod photoreceptors of the mammalian retina. Mutations in NRL are associated with retinopathies; many of these are suggested to change phosphorylation status and alter NRL-mediated transactivation of rhodopsin promoter. The purpose of this study was to identify potential kinases responsible for the phosphorylation of NRL and determine if such kinase-dependent phosphorylation is altered in disease-associated NRL mutations. Methods Metabolic labeling with 33P-orthophosphate was used to study phosphorylation of NRL in transfected COS-1 cells. NRL or NRL mutants were expressed as glutathione S-transferase (GST)-fusion proteins and used as substrate to screen various kinases by in vitro phosphorylation assays. CV-1 cells were co-transfected with rhodopsin promoter-reporter construct and expression plasmids, with or without specific mitogen-activated protein kinase (MAPK) inhibitors, to examine their effect on NRL-mediated transactivation. Expression of activated MAPKs in postnatal mice retina was determined by immunoblot analysis. Results Metabolic labeling of NRL produces multiple phosphorylated protein bands in transfected COS-1 cells. Fewer but more intense radiolabeled bands are observed for NRL-S50T, -S50A, and -P51L mutants compared to wild-type NRL. We show that MAPK2 and p38 induce specific phosphorylation of NRL, but this pattern is altered in NRL mutants. Immunoblot analysis of extracts from developing mouse retina reveals enhanced expression of activated MAPK2 at postnatal day 0-3, concordant with the reported phosphorylation pattern of NRL in vivo. Inhibition of MAPK signaling pathways decreases NRL and CRX -mediated synergistic activation of rhodopsin promoter in transfected CV-1 cells. Conclusions Our results suggest that multiple MAPKs can phosphorylate NRL and this phosphorylation pattern is altered by

  8. Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells.

    PubMed

    Graham, A; McLees, A; Kennedy, C; Gould, G W; Plevin, R

    1996-03-01

    1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor. PMID:8882634

  9. Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells.

    PubMed Central

    Graham, A.; McLees, A.; Kennedy, C.; Gould, G. W.; Plevin, R.

    1996-01-01

    1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8882634

  10. Repeated preconditioning with hyperbaric oxygen induces neuroprotection against forebrain ischemia via suppression of p38 mitogen activated protein kinase.

    PubMed

    Yamashita, Satoshi; Hirata, Takao; Mizukami, Yoichi; Cui, Ying Jun; Fukuda, Shiro; Ishida, Kazuyoshi; Matsumoto, Mishiya; Sakabe, Takefumi

    2009-12-01

    We previously reported in rats that preconditioning with hyperbaric oxygen (HBO; 100% O(2) 3.5-atomsphere absolute (ATA), 1 h/day for 5 days) provided neuroprotection against transient (8 min) forebrain ischemia possibly through protein synthesis relevant to neurotrophin receptor and inflammatory-immune system. A recent report suggested that HBO-induced neuroprotection is relevant to brain derived neurotrophic factor and its downstream event involving suppression of p38 mitogen activated protein kinase (p38) activation. In the present study, we first performed a dose comparison (1, 2, and 3.5 ATA) of HBO-induced neuroprotection and then investigated pharmacological modification by 10 mg/kg anisomycin (a protein synthesis inhibitor and potent activator for p38) and 200 microg/kg SB203580 (a p38 inhibitor), which were given intraperitoneally 60 and 30 min before every 3.5 ATA-HBO treatment, respectively. Most prominent protective effect on hippocampal CA1 neurons was observed with 3.5 ATA-HBO (survived neurons: 69% [62-73%] vs. untreated: 3.9% [2-8%], 1 ATA: 8.8% [0-26%], 2 ATA-HBO: 46% [22-62%] (median [range]) (7 days after ischemia). Anisomycin abolished a neuroprotective effect (survived neuron: 1.2% [0-7%]). SB203580, when given between administration of anisomycin and HBO treatment, resumed a neuroprotective effect (survived neuron: 52% [37-62%]). The level of phosphorylated p38 at 10-min reperfusion was significantly decreased in 3.5 ATA-HBO group (32% [12-53%] of sham). Single pretreatment with 100 and 200 microg/kg of SB203580 exerted a similar neuroprotective effect (39% [25-51%] and 59% [50-72%]) to 2 and 3.5 ATA-HBO preconditioning, respectively. It is concluded that suppression of p38 phosphorylation plays a key role in HBO-induced neuroprotection and that pretreatment with a p38 inhibitor (SB203580) can provide similar neuroprotection. PMID:19747454

  11. Cadmium-induced apoptosis and necrosis in human osteoblasts: role of caspases and mitogen-activated protein kinases pathways.

    PubMed

    Brama, M; Politi, L; Santini, P; Migliaccio, S; Scandurra, R

    2012-02-01

    Cadmium is a widespread environmental pollutant which induces severe toxic alterations, including osteomalacia and osteoporosis, likely by estrogen receptor-dependent mechanisms. Indeed, cadmium has been described to act as an endocrine disruptor and its toxicity is exerted both in vivo and in vitro through induction of apoptosis and/or necrosis by not fully clarified intracellular mechanism(s) of action. Aim of the present study was to further investigate the molecular mechanism by which cadmium might alter homeostasis of estrogen target cells, such as osteoblast homeostasis, inducing cell apoptosis and/or necrosis. Human osteoblastic cells (hFOB 1.19) in culture were used as an in vitro model to characterize the intracellular mechanisms induced by this heavy metal. Cells were incubated in the presence/ absence of 10-50 μM cadmium chloride at different times and DNA fragmentation and activation of procaspases- 8 and -3 were induced upon CdCl(2) treatment triggering apoptotic and necrotic pathways. Addition of caspase-8 and -3 inhibitors (Z-IETD-FMK and Z-DQMD-FMK) partially blocked these effects. No activation of procaspase-9 was observed. To determine the role of mitogen-activated protein kinases (MAPK) in these events, we investigated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated protein kinase (ERK1/2) phosphorylation which were activated by 10 μM CdCl(2). Chemical inhibitors of JNK, p38, and ERK1/2, SP600125, SB202190, and PD98059, significantly reduced the phosphorylation of the kinases and blunted apoptosis. In contrast, caspase inhibitors did not reduce the cadmium-induced MAPK phosphorylation, suggesting an independent activation of these pathways. In conclusion, at least 2 pathways appear activated by cadmium in osteoblasts: a direct induction of caspase-8 followed by activation of caspase-3 and an indirect induction by phosphorylation of ERK1/2, p38, and JNK MAPK triggering activation of caspase-8 and -3. PMID:21697648

  12. Mitogen-activated Protein Kinase Phosphatase-1 Modulates Regional Effects of Injurious Mechanical Ventilation in Rodent Lungs

    PubMed Central

    Park, Moo Suk; Edwards, Michael G.; Sergew, Amen; Riches, David W. H.; Albert, Richard K.

    2012-01-01

    Rationale: Mechanical ventilation induces heterogeneous lung injury by mitogen-activated protein kinase (MAPK) and nuclear factor-κB. Mechanisms regulating regional injury and protective effects of prone positioning are unclear. Objectives: To determine the key regulators of the lung regional protective effects of prone positioning in rodent lungs exposed to injurious ventilation. Methods: Adult rats were ventilated with high (18 ml/kg, positive end-expiratory pressure [PEEP] 0) or low Vt (6 ml/kg; PEEP 3 cm H2O; 3 h) in supine or prone position. Dorsal–caudal lung mRNA was analyzed by microarray and MAPK phosphatases (MKP)-1 quantitative polymerase chain reaction. MKP-1−/− or wild-type mice were ventilated with very high (24 ml/kg; PEEP 0) or low Vt (6–7 ml/kg; PEEP 3 cm H2O). The MKP-1 regulator PG490-88 (MRx-108; 0.75 mg/kg) or phosphate-buffered saline was administered preventilation. Injury was assessed by lung mechanics, bronchioalveolar lavage cell counts, protein content, and lung injury scoring. Immunoblotting for MKP-1, and IκBα and cytokine ELISAs were performed on lung lysates. Measurements and Main Results: Prone positioning was protective against injurious ventilation in rats. Expression profiling demonstrated MKP-1 20-fold higher in rats ventilated prone rather than supine and regional reduction in p38 and c-jun N-terminal kinase activation. MKP-1−/− mice experienced amplified injury. PG490-88 improved static lung compliance and injury scores, reduced bronchioalveolar lavage cell counts and cytokine levels, and induced MKP-1 and IκBα. Conclusions: Injurious ventilation induces MAPK in an MKP-1–dependent fashion. Prone positioning is protective and induces MKP-1. PG490-88 induced MKP-1 and was protective against high Vt in a nuclear factor-κB–dependent manner. MKP-1 is a potential target for modulating regional effects of injurious ventilation. PMID:22582160

  13. Changes in mitogen-activated protein kinase in cerebellar granule neurons by polybrominated diphenyl ethers and polychlorinated biphenyls

    SciTech Connect

    Fan Chunyang; Besas, Jonathan

    2010-05-15

    Polybrominated diphenyl ethers (PBDEs) are used as additive flame retardants and have been detected in human blood, adipose tissue, and breast milk. Both in vitro and in vivo studies have shown that the effects of PBDEs are similar to the known human developmental neurotoxicants such as polychlorinated biphenyls (PCBs) on a molar basis. Previously, we reported that PBDE mixtures and congeners, perturbed calcium homeostasis which is critical for the development and function of the nervous system. In the present study, we tested whether environmentally relevant PBDE/PCB mixtures and congeners affected mitogen-activated protein kinase (MAPK) pathways, which are down-stream events of calcium signaling in cerebellar granule neuronal cultures. In this study, phosphorylated extracellular signal-regulated kinase (pERK)1/2, a widely studied MAPK cascade and known to be involved in learning and memory, levels were quantitated using western blot technique with phospho-specific antibodies. Glutamate (a positive control) increased pERK1/2 in a time- and concentration-dependent manner reaching maximum activation at 5-30 min of exposure and at doses >= 10 muM. Both Aroclor 1254 (a commercial penta PCB mixture) and DE-71 (a commercial penta PBDE mixture) elevated phospho-ERK1/2, producing maximum stimulation at 30 min and at concentrations >= 3 mug/ml; Aroclor 1254 was more efficacious than DE-71. DE-79 (an octabrominated diphenyl ether mixture) also elevated phospho-ERK1/2, but to a lesser extent than that of DE-71. PBDE congeners 47, 77, 99, and 153 also increased phospo-ERK1/2 in a concentration-dependent manner. The data indicated that PBDE congeners are more potent than the commercial mixtures. PCB 47 also increased phospho-ERK1/2 like its structural analog PBDE 47, but to a lesser extent, suggesting that these chemicals affect similar pathways. Cytotoxicity, measured as %LDH release, data showed that higher concentrations (> 30 muM) and longer exposures (> 30 min) are

  14. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

    PubMed Central

    King, W G; Mattaliano, M D; Chan, T O; Tsichlis, P N; Brugge, J S

    1997-01-01

    Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases. PMID:9234699

  15. Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases.

    PubMed Central

    Belham, C M; Tate, R J; Scott, P H; Pemberton, A D; Miller, H R; Wadsworth, R M; Gould, G W; Plevin, R

    1996-01-01

    We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species. PMID:9003384

  16. Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia

    PubMed Central

    Chialda, Ligia; Zhang, Meixia; Brune, Kay; Pahl, Andreas

    2005-01-01

    Background T cells play a dominant role in the pathogenesis of asthma. Costimulation of T cells is necessary to fully activate them. An inducible costimulator (ICOS) of T cells is predominantly expressed on Th2 cells. Therefore, interference of signaling pathways precipitated by ICOS may present new therapeutic options for Th2 dominated diseases such as asthma. However, these signaling pathways are poorly characterized in vitro and in vivo. Methods Human primary CD4+ T cells from blood were activated by beads with defined combinations of surface receptor stimulating antibodies and costimulatory receptor ligands. Real-time RT-PCR was used for measuring the production of cytokines from activated T cells. Activation of mitogen activated protein kinase (MAPK) signaling pathways leading to cytokine synthesis were investigated by western blot analysis and by specific inhibitors. The effect of inhibitors in vivo was tested in a murine asthma model of late phase eosinophilia. Lung inflammation was assessed by differential cell count of the bronchoalveolar lavage, determination of serum IgE and lung histology. Results We showed in vitro that ICOS and CD28 are stimulatory members of an expanding family of co-receptors, whereas PD1 ligands failed to co-stimulate T cells. ICOS and CD28 activated different MAPK signaling cascades necessary for cytokine activation. By means of specific inhibitors we showed that p38 and ERK act downstream of CD28 and that ERK and JNK act downstream of ICOS leading to the induction of various T cell derived cytokines. Using a murine asthma model of late phase eosinophilia, we demonstrated that the ERK inhibitor U0126 and the JNK inhibitor SP600125 inhibited lung inflammation in vivo. This inhibition correlated with the inhibition of Th2 cytokines in the BAL fluid. Despite acting on different signaling cascades, we could not detect synergistic action of any combination of MAPK inhibitors. In contrast, we found that the p38 inhibitor SB203580

  17. Distinct Signaling Properties of Mitogen-activated Protein Kinase Kinases 4 (MKK4) and 7 (MKK7) in Embryonic Stem Cell (ESC) Differentiation*

    PubMed Central

    Wang, Jingcai; Chen, Liang; Ko, Chia-I; Zhang, Lin; Puga, Alvaro; Xia, Ying

    2012-01-01

    Signal transduction pathways are integral components of the developmental regulatory network that guides progressive cell fate determination. MKK4 and MKK7 are upstream kinases of the mitogen-activated protein kinases (MAPKs), responsible for channeling physiological and environmental signals to their cellular responses. Both kinases are essential for survival of mouse embryos, but because of embryonic lethality, their precise developmental roles remain largely unknown. Using gene knock-out mouse ESCs, we studied the roles of MKK4 and MKK7 in differentiation in vitro. While MKK4 and MKK7 were dispensable for ESC self-renewal and pluripotency maintenance, they exhibited unique signaling and functional properties in differentiation. MKK4 and MKK7 complemented each other in activation of the JNK-c-Jun cascades and loss of both led to senescence upon cell differentiation. On the other hand, MKK4 and MKK7 had opposite effects on activation of the p38 cascades during differentiation. Specifically, MKK7 reduced p38 activation, while Mkk7(−/−) ESCs had elevated phosphorylation of MKK4, p38, and ATF2, and increased MEF2C expression. Consequently, Mkk7(−/−) ESCs had higher expression of MHC and MLC and enhanced formation of contractile cardiomyocytes. In contrast, MKK4 was required for p38 activation and Mkk4(−/−) ESCs exhibited diminished p-ATF2 and MEF2C expression, resulting in impaired MHC induction and defective cardiomyocyte differentiation. Exogenous MKK4 expression partially restored the ability of Mkk4(−/−) ESCs to differentiate into cardiomyocytes. Our results uncover complementary and interdependent roles of MKK4 and MKK7 in development, and identify the essential requirement for MKK4 in p38 activation and cardiomyocyte differentiation. PMID:22130668

  18. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

    PubMed Central

    Zinck, R; Cahill, M A; Kracht, M; Sachsenmaier, C; Hipskind, R A; Nordheim, A

    1995-01-01

    Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction. PMID:7651411

  19. 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

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

    PubMed

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

    2015-01-01

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

  1. Activation of group IV cytosolic phospholipase A2 in human eosinophils by phosphoinositide 3-kinase through a mitogen-activated protein kinase-independent pathway.

    PubMed

    Myou, Shigeharu; Leff, Alan R; Myo, Saori; Boetticher, Evan; Meliton, Angelo Y; Lambertino, Anissa T; Liu, Jie; Xu, Chang; Munoz, Nilda M; Zhu, Xiangdong

    2003-10-15

    Activation of group IV cytosolic phospholipase A(2) (gIV-PLA(2)) is the essential first step in the synthesis of inflammatory eicosanoids and in integrin-mediated adhesion of leukocytes. Prior investigations have demonstrated that phosphorylation of gIV-PLA(2) results from activation of at least two isoforms of mitogen-activated protein kinase (MAPK). We investigated the potential role of phosphoinositide 3-kinase (PI3K) in the activation of gIV-PLA(2) and the hydrolysis of membrane phosphatidylcholine in fMLP-stimulated human blood eosinophils. Transduction into eosinophils of Deltap85, a dominant negative form of class IA PI3K adaptor subunit, fused to an HIV-TAT protein transduction domain (TAT-Deltap85) concentration dependently inhibited fMLP-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. FMLP caused increased arachidonic acid (AA) release and secretion of leukotriene C(4) (LTC(4)). TAT-Deltap85 and LY294002, a PI3K inhibitor, blocked the phosphorylation of gIV-PLA(2) at Ser(505) caused by fMLP, thus inhibiting gIV-PLA(2) hydrolysis and production of AA and LTC(4) in eosinophils. FMLP also caused extracellular signal-related kinases 1 and 2 and p38 MAPK phosphorylation in eosinophils; however, neither phosphorylation of extracellular signal-related kinases 1 and 2 nor p38 was inhibited by TAT-Deltap85 or LY294002. Inhibition of 1) p70 S6 kinase by rapamycin, 2) protein kinase B by Akt inhibitor, or 3) protein kinase C by Ro-31-8220, the potential downstream targets of PI3K for activation of gIV-PLA(2), had no effect on AA release or LTC(4) secretion caused by fMLP. We find that PI3K is required for gIV-PLA(2) activation and hydrolytic production of AA in activated eosinophils. Our data suggest that this essential PI3K independently activates gIV-PLA(2) through a pathway that does not involve MAPK. PMID:14530366

  2. Activation of p38 mitogen-activated protein kinase and nuclear factor-kappaB in tumour necrosis factor-induced eotaxin release of human eosinophils

    PubMed Central

    WONG, C K; ZHANG, J P; IP, W K; LAM, C W K

    2002-01-01

    The CC chemokine eotaxin is a potent eosinophil-specific chemoattractant that is crucial for allergic inflammation. Allergen-induced tumour necrosis factor (TNF) has been shown to induce eotaxin synthesis in eosinophils. Nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPK) have been found to play an essential role for the eotaxin-mediated eosinophilia. We investigated the modulation of NF-κB and MAPK activation in TNF-induced eotaxin release of human eosinophils. Human blood eosinophils were purified from fresh buffy coat using magnetic cell sorting. NF-κB pathway-related genes were evaluated by cDNA expression array system. Degradation of IκBα and phosphorylation of MAPK were detected by Western blot. Activation of NF-κB was determined by electrophoretic mobility shift assay. Eotaxin released into the eosinophil culture medium was measured by ELISA. TNF was found to up-regulate the gene expression of NF-κB and IκBα in eosinophils. TNF-induced IκBα degradation was inhibited by the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132) and a non-steroidal anti-inflammatory drug sodium salicylate (NaSal). Using EMSA, both MG-132 and NaSal were found to suppress the TNF-induced NF-κB activation in eosinophils. Furthermore, TNF was shown to induce phosphorylation of p38 MAPK time-dependently but not extracellular signal-regulated kinases (ERK). Inhibition of NF-κB activation and p38 MAPK activity decreased the TNF-induced release of eotaxin from eosinophils. These results indicate that NF-κB and p38 MAPK play an important role in TNF-activated signalling pathway regulating eotaxin release by eosinophils. They have also provided a biochemical basis for the potential of using specific inhibitors of NF-κB and p38 MAPK for treating allergic inflammation. PMID:12067303

  3. Colletotrichum higginsianum Mitogen-Activated Protein Kinase ChMK1: Role in Growth, Cell Wall Integrity, Colony Melanization, and Pathogenicity.

    PubMed

    Wei, Wei; Xiong, Ying; Zhu, Wenjun; Wang, Nancong; Yang, Guogen; Peng, Fang

    2016-01-01

    Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. To facilitate the efficient control of anthracnose disease, it will be important to understand the mechanism by which the cruciferous crops and C. higginsianum interact. A key step in understanding this interaction is characterizing the mitogen-activated protein kinases (MAPK) signaling pathway of C. higginsianum. MAPK plays important roles in diverse physiological processes of multiple pathogens. In this study, a Fus3/Kss1-related MAPK gene, ChMK1, from C. higginsianum was analyzed. The results showed that the Fus3/Kss1-related MAPK ChMK1 plays a significant role in cell wall integrity. Targeted deletion of ChMK1 resulted in a hypersensitivity to cell wall inhibitors, reduced conidiation and albinistic colonies. Further, the deletion mutant was also unable to form melanized appressorium, a specialized infection structure that is necessary for successful infection. Therefore, the deletion mutant loses pathogenicity on A. thaliana leaves, demonstrating that ChMK1 plays an essential role in the early infection step. In addition, the ChMK1 deletion mutant showed an attenuated growth rate that is different from that of its homolog in Colletotrichum lagenarium, indicating the diverse roles that Fus3/Kss1-related MAPKs plays in phytopathogenic fungi. Furthermore, the expression level of three melanin synthesis associated genes were clearly decreased in the albinistic ChMK1 mutant compared to that of the wild type strain, suggesting that ChMK1 is also required for colony melanization in C. higginsianum. PMID:27536296

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

    PubMed

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

    2014-05-01

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

  5. Colletotrichum higginsianum Mitogen-Activated Protein Kinase ChMK1: Role in Growth, Cell Wall Integrity, Colony Melanization, and Pathogenicity

    PubMed Central

    Wei, Wei; Xiong, Ying; Zhu, Wenjun; Wang, Nancong; Yang, Guogen; Peng, Fang

    2016-01-01

    Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. To facilitate the efficient control of anthracnose disease, it will be important to understand the mechanism by which the cruciferous crops and C. higginsianum interact. A key step in understanding this interaction is characterizing the mitogen-activated protein kinases (MAPK) signaling pathway of C. higginsianum. MAPK plays important roles in diverse physiological processes of multiple pathogens. In this study, a Fus3/Kss1-related MAPK gene, ChMK1, from C. higginsianum was analyzed. The results showed that the Fus3/Kss1-related MAPK ChMK1 plays a significant role in cell wall integrity. Targeted deletion of ChMK1 resulted in a hypersensitivity to cell wall inhibitors, reduced conidiation and albinistic colonies. Further, the deletion mutant was also unable to form melanized appressorium, a specialized infection structure that is necessary for successful infection. Therefore, the deletion mutant loses pathogenicity on A. thaliana leaves, demonstrating that ChMK1 plays an essential role in the early infection step. In addition, the ChMK1 deletion mutant showed an attenuated growth rate that is different from that of its homolog in Colletotrichum lagenarium, indicating the diverse roles that Fus3/Kss1-related MAPKs plays in phytopathogenic fungi. Furthermore, the expression level of three melanin synthesis associated genes were clearly decreased in the albinistic ChMK1 mutant compared to that of the wild type strain, suggesting that ChMK1 is also required for colony melanization in C. higginsianum. PMID:27536296

  6. Structural characterization and immunomodulatory activity of Grifola frondosa polysaccharide via toll-like receptor 4-mitogen-activated protein kinases-nuclear factor κB pathways.

    PubMed

    Ma, Xiaolei; Meng, Meng; Han, Lirong; Cheng, Dai; Cao, Xiaohong; Wang, Chunling

    2016-06-15

    We isolated a neutral polysaccharide from the fruiting body of a mushroom Grifola frondosa (GFP-A). The aim of this study was to characterize a neutral α-d-polysaccharide derived from G. frondosa and evaluate its immunomodulatory effect on toll-like receptor 4, mitogen-activated protein kinases and nuclear factor κB pathways of protein expression in macrophages. The structural features of GFP-A were characterized by physicochemical and instrumental analyses. Its molecular weight was found to be 8.48 × 10(2) kDa. The main chain of GFP-A consisted of (1 → 4)-linked and (1 → 6)-linked α-d-glucopyranosyl, and (1 → 3,6)-linked α-d-mannopyranosyl residues, which branched at C-3. The branches consisted of (1 → 6)-linked α-d-galactopyranosyl and t-l-rhamnopyranosyl residues. An in vitro immunomodulatory assay for pro-inflammatory cytokines (interleukin-1β, interleukin-2, tumor necrosis factor alpha, etc.) using the macrophage cell line, RAW 264.7, revealed that GFP-A exhibited significant immunomodulatory activity by stimulating the toll-like receptor 4, mitogen-activated protein kinases to nuclear factor κB/pathway. PMID:27220562

  7. Bacterial AvrRpt2-Like Cysteine Proteases Block Activation of the Arabidopsis Mitogen-Activated Protein Kinases, MPK4 and MPK111[OPEN

    PubMed Central

    Eschen-Lippold, Lennart; Jiang, Xiyuan; Elmore, James Mitch; Mackey, David; Shan, Libo

    2016-01-01

    To establish infection, pathogens deliver effectors into host cells to target immune signaling components, including elements of mitogen-activated protein kinase (MPK) cascades. The virulence function of AvrRpt2, one of the first identified Pseudomonas syringae effectors, involves cleavage of the plant defense regulator, RPM1-INTERACTING PROTEIN4 (RIN4), and interference with plant auxin signaling. We show now that AvrRpt2 specifically suppresses the flagellin-induced phosphorylation of Arabidopsis (Arabidopsis thaliana) MPK4 and MPK11 but not MPK3 or MPK6. This inhibition requires the proteolytic activity of AvrRpt2, is associated with reduced expression of some plant defense genes, and correlates with enhanced pathogen infection in AvrRpt2-expressing transgenic plants. Diverse AvrRpt2-like homologs can be found in some phytopathogens, plant-associated and soil bacteria. Employing these putative bacterial AvrRpt2 homologs and inactive AvrRpt2 variants, we can uncouple the inhibition of MPK4/MPK11 activation from the cleavage of RIN4 and related members from the so-called nitrate-induced family as well as from auxin signaling. Thus, this selective suppression of specific mitogen-activated protein kinases is independent of the previously known AvrRpt2 targets and potentially represents a novel virulence function of AvrRpt2. PMID:27208280

  8. Bacterial AvrRpt2-Like Cysteine Proteases Block Activation of the Arabidopsis Mitogen-Activated Protein Kinases, MPK4 and MPK11.

    PubMed

    Eschen-Lippold, Lennart; Jiang, Xiyuan; Elmore, James Mitch; Mackey, David; Shan, Libo; Coaker, Gitta; Scheel, Dierk; Lee, Justin

    2016-07-01

    To establish infection, pathogens deliver effectors into host cells to target immune signaling components, including elements of mitogen-activated protein kinase (MPK) cascades. The virulence function of AvrRpt2, one of the first identified Pseudomonas syringae effectors, involves cleavage of the plant defense regulator, RPM1-INTERACTING PROTEIN4 (RIN4), and interference with plant auxin signaling. We show now that AvrRpt2 specifically suppresses the flagellin-induced phosphorylation of Arabidopsis (Arabidopsis thaliana) MPK4 and MPK11 but not MPK3 or MPK6. This inhibition requires the proteolytic activity of AvrRpt2, is associated with reduced expression of some plant defense genes, and correlates with enhanced pathogen infection in AvrRpt2-expressing transgenic plants. Diverse AvrRpt2-like homologs can be found in some phytopathogens, plant-associated and soil bacteria. Employing these putative bacterial AvrRpt2 homologs and inactive AvrRpt2 variants, we can uncouple the inhibition of MPK4/MPK11 activation from the cleavage of RIN4 and related members from the so-called nitrate-induced family as well as from auxin signaling. Thus, this selective suppression of specific mitogen-activated protein kinases is independent of the previously known AvrRpt2 targets and potentially represents a novel virulence function of AvrRpt2. PMID:27208280

  9. Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxy)benzoic Acid

    PubMed Central

    Lee, Kuan-Han; Ho, Wen-Yueh; Wu, Shu-Jing; Omar, Hany A.; Huang, Po-Jui; Wang, Clay C. C.; Hung, Jui-Hsiang

    2014-01-01

    Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxy)benzoic acid (TMPBA) and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 μM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP) kinases, 5′ adenosine monophosphate-activated protein kinase (AMPK), and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK) signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy. PMID:24406729

  10. GhMPK17, a Cotton Mitogen-Activated Protein Kinase, Is Involved in Plant Response to High Salinity and Osmotic Stresses and ABA Signaling

    PubMed Central

    Li, Yang; Sun, Xiang; Wang, Na-Na; Gong, Si-Ying; Zheng, Yong; Li, Xue-Bao

    2014-01-01

    Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. Cotton (Gossypium hirsutum) is the most important textile crop in the world, and often encounters abiotic stress during its growth seasons. In this study, a gene encoding a mitogen-activated protein kinase (MAPK) was isolated from cotton, and designated as GhMPK17. The open reading frame (ORF) of GhMPK17 gene is 1494 bp in length and encodes a protein with 497 amino acids. Quantitative RT-PCR analysis indicated that GhMPK17 expression was up-regulated in cotton under NaCl, mannitol and ABA treatments. The transgenic Arabidopsis plants expressing GhMPK17 gene showed higher seed germination, root elongation and cotyledon greening/expansion rates than those of the wild type on MS medium containing NaCl, mannitol and exogenous ABA, suggesting that overexpression of GhMPK17 in Arabidopsis increased plant ABA-insensitivity, and enhanced plant tolerance to salt and osmotic stresses. Furthermore, overexpression of GhMPK17 in Arabidopsis reduced H2O2 level and altered expression of ABA- and abiotic stress-related genes in the transgenic plants. Collectively, these data suggested that GhMPK17 gene may be involved in plant response to high salinity and osmotic stresses and ABA signaling. PMID:24743296

  11. Interaction of nuclear protein p140 with human immunodeficiency virus type 1 TAR RNA in mitogen-activated primary human T lymphocytes.

    PubMed Central

    Rothblum, C J; Jackman, J; Mikovits, J; Shukla, R R; Kumar, A

    1995-01-01

    Several lines of evidence suggest that cellular proteins play a role during human immunodeficiency virus type 1 (HIV-1) Tat-mediated trans activation. A recent report from this laboratory has shown that a 140-kDa HeLa nuclear protein (p140) binds specifically to the lower stem region of the Tat response element, TAR RNA. Since HIV-1 trans activation is most efficient in proliferating T cells, we investigated the binding of p140 to TAR RNA in unstimulated and mitogen-activated, G1-phase primary T lymphocytes. TAR RNA/protein-binding activity was low in resting cells but increased significantly within 2 h of activation and remained elevated for at least 48 h. Corresponding increases in p140 protein levels were observed with most but not all donors, suggesting that an additional nuclear factor(s) may be required for efficient binding of this protein to TAR RNA in activated T cells. PMID:7609087

  12. Role of mitogen-activated protein kinases and nuclear factor-kappa B in 1,3-dichloro-2-propanol-induced hepatic injury

    PubMed Central

    Lee, In-Chul; Lee, Sang-Min; Ko, Je-Won; Park, Sung-Hyeuk; Shin, In-Sik; Moon, Changjong; Kim, Sung-Ho

    2016-01-01

    In this study, the potential hepatotoxicity of 1,3-dichloro-2-propanol and its hepatotoxic mechanisms in rats was investigated. The test chemical was administered orally to male rats at 0, 27.5, 55, and 110 mg/kg body weight. 1,3-Dichloro-2-propanol administration caused acute hepatotoxicity, as evidenced by an increase in serum aminotransferases, total cholesterol, and total bilirubin levels and a decrease in serum glucose concentration in a dose-dependent manner with corresponding histopathological changes in the hepatic tissues. The significant increase in malondialdehyde content and the significant decrease in glutathione content and antioxidant enzyme activities indicated that 1,3-dichloro-2-propanol-induced hepatic damage was mediated through oxidative stress, which caused a dose-dependent increase of hepatocellular apoptotic changes in the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and immunohistochemical analysis for caspase-3. The phosphorylation of mitogen-activated protein kinases caused by 1,3-dichloro-2-propanol possibly involved in hepatocellular apoptotic changes in rat liver. Furthermore, 1,3-dichloro-2-propanol induced an inflammatory response through activation of nuclear factor-kappa B signaling that coincided with the induction of pro-inflammatory mediators or cytokines in a dose-dependent manner. Taken together, these results demonstrate that hepatotoxicity may be related to oxidative stress-mediated activation of mitogen-activated protein kinases and nuclear factor-kappa B-mediated inflammatory response. PMID:27051440

  13. Activated protein C downregulates p38 mitogen-activated protein kinase and improves clinical parameters in an in-vivo model of septic shock.

    PubMed

    Nold, Marcel F; Nold-Petry, Claudia A; Fischer, Doris; Richter, Bernd; Blaheta, Roman; Pfeilschifter, Josef; Muhl, Heiko; Schranz, Dietmar; Veldman, Alex

    2007-11-01

    Despite the success of the anti-coagulant protease protein C (PC) in treating septic shock in humans, the signaling pathways used are still unclear. To explore the effects of treatment with PC zymogen and its activated form aPC in a setting of sepsis, we employed a piglet model of endotoxic shock. In the aPC group, we observed a 65%-90% reduction in plasma TNF-alpha levels and a concomitant clinical improvement. Unexpectedly, administration of aPC also resulted in stabilization of the plasma pH above 7.2. Moreover, phosphorylated p38 mitogen-activated protein kinase (p38MAPK) was virtually absent in the livers of those piglets receiving aPC. In cultured human umbilical vein endothelial cells, we observed that nanomolar concentrations of PC and aPC inhibited the phosphorylation of p38MAPK. Furthermore, we showed that the regulation of the pro-apoptotic cell cycle regulator p53 by PC and aPC is dependent on the reduction of p38MAPK activation. The transduction of these effects involves all three receptors associated with protein C signaling, namely endothelial protein C receptor, protease-activated receptor 1, and sphingosine 1-phosphate receptor 1. Ultimately, this study elucidates novel signaling pathways regulated by protein C and emphasises the pivotal importance of its multiple modes of action beyond anticoagulation. APC's clinical success may, in part, be due to p38MAPK inhibition. PMID:18000619

  14. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    SciTech Connect

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  15. Role of the Unfolded Protein Response in Regulating the Mucin-Dependent Filamentous-Growth Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Adhikari, Hema; Vadaie, Nadia; Chow, Jacky; Caccamise, Lauren M.; Chavel, Colin A.; Li, Boyang; Bowitch, Alexander; Stefan, Christopher J.

    2015-01-01

    Signaling mucins are evolutionarily conserved regulators of signal transduction pathways. The signaling mucin Msb2p regulates the Cdc42p-dependent mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in yeast. The cleavage and release of the glycosylated inhibitory domain of Msb2p is required for MAPK activation. We show here that proteolytic processing of Msb2p was induced by underglycosylation of its extracellular domain. Cleavage of underglycosylated Msb2p required the unfolded protein response (UPR), a quality control (QC) pathway that operates in the endoplasmic reticulum (ER). The UPR regulator Ire1p, which detects misfolded/underglycosylated proteins in the ER, controlled Msb2p cleavage by regulating transcriptional induction of Yps1p, the major protease that processes Msb2p. Accordingly, the UPR was required for differentiation to the filamentous cell type. Cleavage of Msb2p occurred in conditional trafficking mutants that trap secretory cargo in the endomembrane system. Processed Msb2p was delivered to the plasma membrane, and its turnover by the ubiquitin ligase Rsp5p and ESCRT attenuated the filamentous-growth pathway. We speculate that the QC pathways broadly regulate signaling glycoproteins and their cognate pathways by recognizing altered glycosylation patterns that can occur in response to extrinsic cues. PMID:25666509

  16. Effect of p38 mitogen-activate protein kinase on MUC5AC protein expression of bile duct epithelial cells in hepatolithiasis patients

    PubMed Central

    Wang, Ping; Ma, Xiaodong; He, Yu; Sun, Beiwang; Zhu, Canhua; Zhao, Rujin; Zhang, Shaoling; Huang, Xianxian; Liu, Yanmin

    2015-01-01

    Primary hepatolithiasis is a common bile duct disease with benign nature but complicated mechanisms. Current studies have revealed its correlation with cytokine release by chronic inflammation, which also increased mucin (MUC) synthesis. This study investigated the role of p38 mitogen-activated protein kinase (MAPK) in regulating cytokine release and mucin synthesis, in an attempt to elucidate the role of p38 signaling molecule in the pathogenesis of hepatolithiasis. In human intrahepatic bile duct endothelial cells (HIBECs), lipoprotein (LPS) was used to induce the high expression of MUC. Small interference RNA (siRNA) was then used to silencing p38 gene expression. Cytokines including interleukin (IL)-1β and tumor necrosis factor (TNF)-α were measured, along with MUC5AC protein and mRNA expression assay. The interference of p38 gene expression inhibited the release of IL-1β and TNF-α in cultured cells. It also depressed both mRNA and protein levels of MUC5A. P38 MAPK signal pathway may be involved in the formation and progression of hepatolithiasis. This study provides potential new strategy for treating hepatolithiasis using p38 MAPK signal pathway as the drug target. PMID:26722604

  17. Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

    PubMed

    Maisonneuve, Pierre; Caillet-Saguy, Célia; Vaney, Marie-Christine; Bibi-Zainab, Edoo; Sawyer, Kristi; Raynal, Bertrand; Haouz, Ahmed; Delepierre, Muriel; Lafon, Monique; Cordier, Florence; Wolff, Nicolas

    2016-08-01

    The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction. PMID:27246854

  18. Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway

    PubMed Central

    Yang, Jing; Ye, Ling; Hui, Tian-Qian; Yang, Dong-Mei; Huang, Ding-Ming; Zhou, Xue-Dong; Mao, Jeremy J; Wang, Cheng-Lin

    2015-01-01

    Both bone morphogenetic protein 2 (BMP2) and the wingless-type MMTV integration site (WNT)/β-catenin signalling pathway play important roles in odontoblast differentiation and dentinogenesis. Cross-talk between BMP2 and WNT/β-catenin in osteoblast differentiation and bone formation has been identified. However, the roles and mechanisms of the canonical WNT pathway in the regulation of BMP2 in dental pulp injury and repair remain largely unknown. Here, we demonstrate that BMP2 promotes the differentiation of human dental pulp cells (HDPCs) by activating WNT/β-catenin signalling, which is further mediated by p38 mitogen-activated protein kinase (MAPK) in vitro. BMP2 stimulation upregulated the expression of β-catenin in HDPCs, which was abolished by SB203580 but not by Noggin or LDN193189. Furthermore, BMP2 enhanced cell differentiation, which was not fully inhibited by Noggin or LDN193189. Instead, SB203580 partially blocked BMP2-induced β-catenin expression and cell differentiation. Taken together, these data suggest a possible mechanism by which the elevation of β-catenin resulting from BMP2 stimulation is mediated by the p38 MAPK pathway, which sheds light on the molecular mechanisms of BMP2-mediated pulp reparative dentin formation. PMID:26047580

  19. Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice[C][W

    PubMed Central

    Xie, Kabin; Chen, Jianping; Wang, Qin; Yang, Yinong

    2014-01-01

    The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery. PMID:25035404

  20. Stearoyl lysophosphatidylcholine enhances the phagocytic ability of macrophages through the AMP-activated protein kinase/p38 mitogen activated protein kinase pathway.

    PubMed

    Quan, Hui; Hur, Young-Hoe; Xin, Chun; Kim, Joung-Min; Choi, Jeong-Il; Kim, Man-Young; Bae, Hong-Beom

    2016-10-01

    A previous study showed that stearoyl lysophosphatidylcholine (sLPC) suppressed extracellular high mobility group box 1 translocation in macrophages stimulated with lipopolysaccharide through AMP-activated protein kinase (AMPK) activation. In the present study, we investigated whether sLPC-induced AMPK activation could enhance macrophages phagocytosis of bacteria. We found that sLPC increased phosphorylation of AMPK and acetyl-CoA carboxylase, a downstream target of AMPK, in a time- and dose-dependent manner in macrophages. Furthermore, sLPC increased the uptake of FITC-conjugated Escherichia coli by macrophages in a dose-dependent manner, and treatment with an AMPK inhibitor (compound C) or siRNA to AMPKα1 reversed this uptake. sLPC increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK), but inhibition of AMPK activity with compound C or siRNA to AMPKα1 prevented the sLPC-induced increase in p38 MAPK phosphorylation. SB203580, a p38 MAPK inhibitor, decreased sLPC-induced phagocytosis. In vivo, systemic administration of sLPC to mice led to increased AMPK and p38 MAPK activity in the lung and to increased phagocytosis of fluorescent E. coli in bronchoalveolar lavage cells. These results suggest that sLPC increases macrophages phagocytosis through activation of the AMPK/p38 MAPK pathway. Therefore, sLPC is a candidate pharmacological agent for the treatment of bacterial infections in clinically relevant conditions. PMID:27517519

  1. Involvement of YODA and mitogen activated protein kinase 6 in Arabidopsis post-embryogenic root development through auxin up-regulation and cell division plane orientation.

    PubMed

    Smékalová, Veronika; Luptovčiak, Ivan; Komis, George; Šamajová, Olga; Ovečka, Miroslav; Doskočilová, Anna; Takáč, Tomáš; Vadovič, Pavol; Novák, Ondřej; Pechan, Tibor; Ziemann, Anja; Košútová, Petra; Šamaj, Jozef

    2014-09-01

    The role of YODA MITOGEN ACTIVATED PROTEIN KINASE KINASE KINASE 4 (MAPKKK4) upstream of MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) was studied during post-embryonic root development of Arabidopsis thaliana. Loss- and gain-of-function mutants of YODA (yda1 and ΔNyda1) were characterized in terms of root patterning, endogenous auxin content and global proteomes. We surveyed morphological and cellular phenotypes of yda1 and ΔNyda1 mutants suggesting possible involvement of auxin. Endogenous indole-3-acetic acid (IAA) levels were up-regulated in both mutants. Proteomic analysis revealed up-regulation of auxin biosynthetic enzymes tryptophan synthase and nitrilases in these mutants. The expression, abundance and phosphorylation of MPK3, MPK6 and MICROTUBULE ASSOCIATED PROTEIN 65-1 (MAP65-1) were characterized by quantitative polymerase chain reaction (PCR) and western blot analyses and interactions between MAP65-1, microtubules and MPK6 were resolved by quantitative co-localization studies and co-immunoprecipitations. yda1 and ΔNyda1 mutants showed disoriented cell divisions in primary and lateral roots, abortive cytokinesis, and differential subcellular localization of MPK6 and MAP65-1. They also showed deregulated expression of TANGLED1 (TAN1), PHRAGMOPLAST ORIENTING KINESIN 1 (POK1), and GAMMA TUBULIN COMPLEX PROTEIN 4 (GCP4). The findings that MPK6 localized to preprophase bands (PPBs) and phragmoplasts while the mpk6-4 mutant transformed with MPK6AEF (alanine (A)-glutamic acid (E)-phenylanine (F)) showed a root phenotype similar to that of yda1 demonstrated that MPK6 is an important player downstream of YODA. These data indicate that YODA and MPK6 are involved in post-embryonic root development through an auxin-dependent mechanism regulating cell division and mitotic microtubule (PPB and phragmoplast) organization. PMID:24923680

  2. Antimelanogenesis Activity of Hydrolyzed Ginseng Extract (GINST) via Inhibition of JNK Mitogen-activated Protein Kinase in B16F10 Cells.

    PubMed

    Han, Joon-Seung; Sung, Jong Hwan; Lee, Seung Kwon

    2016-08-01

    GINST is a hydrolyzed ginseng extract produced by an in vitro process that imitates the metabolic function of bacteria in the human digestive track and has approved by the Ministry of Food and Drug Safety of Korea for the management of postprandial hyperglycemia. Additionally, GINST has been reported to have other physiological functions including anti-aging and antioxidant effects. The objectives of this study are to compare the antimelanogenic effects of fresh ginseng extract (FGE) and GINST extract and to elucidate the functional mechanism. The concentration of total ginsenosides in FGE and GINST was measured using ultraperformance liquid chromatography with a C18 column. B16F10 cells were treated with FGE and GINST for 72 h to assess melanin content, tyrosinase activity, and protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase-related protein-1 (TRP-1). The activity of kinases involved in mitogen-activated protein kinase (MAPK) signaling, such as extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (p38), were measured using western blots. While neither FGE nor GINST inhibited the activity of mushroom tyrosinase directly, GINST decreased melanogenesis and tyrosinase activity markedly. Furthermore, our results indicate that GINST downregulated the levels of MITF and TRP-1 possibly by suppressing JNK signaling. We concluded that, when compared to FGE, GINST has a superior antimelanogenic effect mediated by the downregulation of MITF, TRP-1, and intracellular tyrosinase activity via the JNK signaling pathway. Thus, we suggest that GINST has the potential to be used as a novel skin whitening agent. PMID:27356239

  3. Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4.

    PubMed Central

    Whalen, S G; Marcellus, R C; Whalen, A; Ahn, N G; Ricciardi, R P; Branton, P E

    1997-01-01

    A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter. PMID:9094626

  4. The Arabidopsis transcription factor BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 is a direct substrate of MITOGEN-ACTIVATED PROTEIN KINASE6 and regulates immunity.

    PubMed

    Kang, Sining; Yang, Fan; Li, Lin; Chen, Huamin; Chen, She; Zhang, Jie

    2015-03-01

    Pathogen-associated molecular patterns (PAMPs) are recognized by plant pattern recognition receptors to activate PAMP-triggered immunity (PTI). Mitogen-activated protein kinases (MAPKs), as well as other cytoplasmic kinases, integrate upstream immune signals and, in turn, dissect PTI signaling via different substrates to regulate defense responses. However, only a few direct substrates of these signaling kinases have been identified. Here, we show that PAMP perception enhances phosphorylation of BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1), a transcription factor involved in brassinosteroid (BR) signaling pathway, through pathogen-induced MAPKs in Arabidopsis (Arabidopsis thaliana). BES1 interacts with MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and is phosphorylated by MPK6. bes1 loss-of-function mutants display compromised resistance to bacterial pathogen Pseudomonas syringae pv tomato DC3000. BES1 S286A/S137A double mutation (BES1(SSAA)) impairs PAMP-induced phosphorylation and fails to restore bacterial resistance in bes1 mutant, indicating a positive role of BES1 phosphorylation in plant immunity. BES1 is phosphorylated by glycogen synthase kinase3 (GSK3)-like kinase BR-insensitive2 (BIN2), a negative regulator of BR signaling. BR perception inhibits BIN2 activity, allowing dephosphorylation of BES1 to regulate plant development. However, BES1(SSAA) does not affect BR-mediated plant growth, suggesting differential residue requirements for the modulation of BES1 phosphorylation in PTI and BR signaling. Our study identifies BES1 as a unique direct substrate of MPK6 in PTI signaling. This finding reveals MAPK-mediated BES1 phosphorylation as another BES1 modulation mechanism in plant cell signaling, in addition to GSK3-like kinase-mediated BES1 phosphorylation and F box protein-mediated BES1 degradation. PMID:25609555

  5. Visualizing and Quantitating the Spatiotemporal Regulation of Ras/ERK Signaling by Dual-Specificity Mitogen-Activated Protein Phosphatases (MKPs).

    PubMed

    Caunt, Christopher J; Kidger, Andrew M; Keyse, Stephen M

    2016-01-01

    The spatiotemporal regulation of the Ras/ERK pathway is critical in determining the physiological and pathophysiological outcome of signaling. Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (DUSPs or MKPs) are key regulators of pathway activity and may also localize ERK to distinct subcellular locations. Here we present methods largely based on the use of high content microscopy to both visualize and quantitate the subcellular distribution of activated (p-ERK) and total ERK in populations of mouse embryonic fibroblasts derived from mice lacking DUSP5, a nuclear ERK-specific MKP. Such methods in combination with rescue experiments using adenoviral vectors encoding wild-type and mutant forms of DUSP5 have allowed us to visualize specific defects in ERK regulation in these cells thus confirming the role of this phosphatase as both a nuclear regulator of ERK activity and localization. PMID:27514808

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

    PubMed

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

    2006-09-01

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

  7. Chlamydiaphage φCPG1 Capsid Protein Vp1 Inhibits Chlamydia trachomatis Growth via the Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Guo, Yuanli; Guo, Rui; Zhou, Quan; Sun, Changgui; Zhang, Xinmei; Liu, Yuanjun; Liu, Quanzhong

    2016-01-01

    Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK) pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL)-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis. PMID:27089359

  8. Chlamydiaphage φCPG1 Capsid Protein Vp1 Inhibits Chlamydia trachomatis Growth via the Mitogen-Activated Protein Kinase Pathway.

    PubMed

    Guo, Yuanli; Guo, Rui; Zhou, Quan; Sun, Changgui; Zhang, Xinmei; Liu, Yuanjun; Liu, Quanzhong

    2016-04-01

    Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK) pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL)-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis. PMID:27089359

  9. Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNF{alpha} in human epidermal keratinocytes (HaCaT)

    SciTech Connect

    Onda, Kenji . E-mail: knjond@ps.toyaku.ac.jp; Nagashima, Masahiro; Kawakubo, Yo; Inoue, Shota; Hirano, Toshihiko; Oka, Kitaro

    2006-12-08

    Glucocorticoids (GCs) are essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) {alpha} reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNF{alpha} was not accompanied by changes in mRNA expressions of GR isoforms ({alpha} or {beta}). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNF{alpha}. Additionally, we observed that TNF{alpha} reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNF{alpha}-mediated GC insensitivity. Our data suggest that overexpression of TNF{alpha} leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.

  10. Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Antagonistically Regulate Fission Yeast fbp1 Transcription by Employing Different Modes of Action at Two Upstream Activation Sites

    PubMed Central

    Neely, Lori A.; Hoffman, Charles S.

    2000-01-01

    A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site. PMID:10938120

  11. p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

    PubMed

    Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T; Arumäe, Urmas; Lindholm, Dan

    2016-05-13

    Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. PMID:26984409

  12. A computational model on the modulation of mitogen-activated protein kinase (MAPK) and Akt pathways in heregulin-induced ErbB signalling.

    PubMed Central

    Hatakeyama, Mariko; Kimura, Shuhei; Naka, Takashi; Kawasaki, Takuji; Yumoto, Noriko; Ichikawa, Mio; Kim, Jae-Hoon; Saito, Kazuki; Saeki, Mihoro; Shirouzu, Mikako; Yokoyama, Shigeyuki; Konagaya, Akihiko

    2003-01-01

    ErbB tyrosine kinase receptors mediate mitogenic signal cascade by binding a variety of ligands and recruiting the different cassettes of adaptor proteins. In the present study, we examined heregulin (HRG)-induced signal transduction of ErbB4 receptor and found that the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway negatively regulated the extracellular signal-regulated kinase (ERK) cascade by phosphorylating Raf-1 on Ser(259). As the time-course kinetics of Akt and ERK activities seemed to be transient and complex, we constructed a mathematical simulation model for HRG-induced ErbB4 receptor signalling to explain the dynamics of the regulation mechanism in this signal transduction cascade. The model reflected well the experimental results observed in HRG-induced ErbB4 cells and in other modes of growth hormone-induced cell signalling that involve Raf-Akt cross-talk. The model suggested that HRG signalling is regulated by protein phosphatase 2A as well as Raf-Akt cross-talk, and protein phosphatase 2A modulates the kinase activity in both the PI3K-Akt and MAPK (mitogen-activated protein kinase) pathways. PMID:12691603

  13. PTH stimulated growth and decreased Col-X deposition are phosphotidylinositol-3,4,5 triphosphate kinase and mitogen activating protein kinase dependent in avian sterna.

    PubMed

    Harrington, Erik Kern; Coon, David J; Kern, Matthew F; Svoboda, Kathy K H

    2010-02-01

    Type X collagen (Col-X) deposition is a marker of terminal differentiation during chondrogenesis, in addition to appositional growth and apoptosis. The parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor, or PPR, is a G-Protein coupled receptor (GPCR), which activates several downstream pathways, moderating chondrocyte differentiation, including suppression of Col-X deposition. An Avian sterna model was used to analyze the PPR GPCR downstream kinase role in growth rate and extracellular matrix (ECM) including Col-II, IX, and X. Phosphatidylinositol kinase (PI3K), mitogen activating protein kinase (MAPK) and protein kinase A (PKA) were inhibited with specific established inhibitors LY294002, PD98059, and H89, respectively to test the hypothesis that they could reverse/inhibit the PTH/PTHrP pathway. Excised E14 chick sterna were PTH treated with or without an inhibitor and compared to controls. Sternal length was measured every 24 hr. Cultured sterna were immuno-stained using specific antibodies for Col-II, IX, or X and examined via confocal microscopy. Increased growth in PTH-treated sterna was MAPK, PI3K, and PKA dose dependent, suggesting growth was regulated through multiple pathways. Col-X deposition was rescued in PTH-treated sterna in the presence of PI3K or MAPK inhibitors, but not with the PKA inhibitor. All three inhibitors moderately disrupted Col-II and Col-IX deposition. These results suggest that PTH can activate multiple pathways during chondrocyte differentiation. PMID:19957341

  14. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed Central

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-01-01

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  15. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-04-15

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  16. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2014-01-01

    Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation. PMID:25421240

  17. Whey protein concentrate enhances intestinal integrity and influences transforming growth factor-β1 and mitogen-activated protein kinase signalling pathways in piglets after lipopolysaccharide challenge.

    PubMed

    Xiao, Kan; Jiao, Lefei; Cao, Shuting; Song, Zehe; Hu, Caihong; Han, Xinyan

    2016-03-28

    attenuates the LPS-induced intestinal injury by improving mucosal barrier function, alleviating intestinal inflammation and influencing TGF-β1 canonical Smad and mitogen-activated protein kinase signalling pathways. PMID:26810899

  18. The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitization.

    PubMed

    Licata, Stephanie C; Pierce, R Christopher

    2003-04-01

    Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization. PMID:12641723

  19. Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

    PubMed Central

    Abrieu, A; Dorée, M; Picard, A

    1997-01-01

    The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation. Images PMID:9190205

  20. Proliferative effects of gamma-amino butyric acid on oral squamous cell carcinoma cells are associated with mitogen-activated protein kinase signaling pathways.

    PubMed

    Ma, Jing; Zhang, Yan; Wang, Jun; Zhao, Tianyu; Ji, Ping; Song, Jinlin; Zhang, Hongmei; Luo, Wenping

    2016-07-01

    Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult mammalian central nervous system, has been reported to play an important physiological role in peripheral non-neuronal tissues, such as tumors. However, whether deregulated GABA is associated with oral squamous cell carcinoma (OSCC) is currently unknown. In this study, we investigated the effects of GABA on the proliferation of the OSCC cell line, Tca8113. Immunohistochemical analyses were performed to examine the expression of GABA A type receptor pi subunit (GABRP) in human OSCC tissues, and reverse transcription polymerase chain reaction, immunofluorescence staining and western blot analysis were performed to examine the expression of GABRP in Tca8113 cells. The proliferative effects of GABA on Tca8113 cells were analyzed by CCK-8 assay and flow cytometry. The activation status of mitogen-activated protein kinases (MAPKs) was examined by western blot analysis. GABRP expression was observed in the cytoplasm with a higher level in poorly differentiated OSCC tissues. The mRNA and protein expression levels of GABRP were detected in the Tca8113 cells. The addition of GABA and the GABA A type receptor agonist, Muscimol, promoted cell proliferation and inhibited cell apoptosis through the activation of the p38 MAPK and the inhibition of the JNK MAPK signaling pathways. These results imply a novel role of GABA in OSCC. PMID:27222045

  1. CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids

    PubMed Central

    2012-01-01

    Background Mitogen activated protein kinase (MAPK) cascade is an important signaling cascade that operates in stress signal transduction in plants. The biologically active monoterpenoid indole alkaloids (MIA) produced in Catharanthus roseus are known to be induced under several abiotic stress conditions such as wounding, UV-B etc. However involvement of any signaling component in the accumulation of MIAs remains poorly investigated so far. Here we report isolation of a novel abiotic stress inducible Catharanthus roseus MAPK, CrMPK3 that may have role in accumulation of MIAs in response to abiotic stress. Results CrMPK3 expressed in bacterial system is an active kinase as it showed auto-phosphorylation and phosphorylation of Myelin Basic Protein. CrMPK3 though localized in cytoplasm, moves to nucleus upon wounding. Wounding, UV treatment and MeJA application on C. roseus leaves resulted in the transcript accumulation of CrMPK3 as well as activation of MAPK in C. roseus leaves. Immuno-precipitation followed by immunoblot analysis revealed that wounding, UV treatment and methyl jasmonate (MeJA) activate CrMPK3. Transient over-expression of CrMPK3 in C. roseus leaf tissue showed enhanced expression of key MIA biosynthesis pathway genes and also accumulation of specific MIAs. Conclusion Results from our study suggest a possible involvement of CrMPK3 in abiotic stress signal transduction towards regulation of transcripts of key MIA biosynthetic pathway genes, regulators and accumulation of major MIAs. PMID:22871174

  2. Dual role for mitogen-activated protein kinase (Erk) in insulin-dependent regulation of Fra-1 (fos-related antigen-1) transcription and phosphorylation.

    PubMed Central

    Hurd, Toby W; Culbert, Ainsley A; Webster, Kenneth J; Tavaré, Jeremy M

    2002-01-01

    Insulin regulates the activity of the AP-1 (activator protein-1) transcriptional complex in several cell types. One component of the AP-1 complex is the transcription factor Fra-1 (fos-related antigen-1), and we have demonstrated previously that insulin stimulates the expression of Fra-1 mRNA in CHO.T cells [Griffiths, Black, Culbert, Dickens, Shaw, Gillespie and Tavaré (1998) Biochem. J. 335, 19-26]. Here we demonstrate that insulin stimulates the activity of a fra-1 promoter linked to a luciferase reporter gene, indicating that the ability of insulin to induce expression of Fra-1 mRNA is due, at least in part, to an increase in gene transcription. Furthermore, we found that insulin induces the serine phosphorylation of Fra-1 and reduces its mobility during SDS/PAGE as a result of phosphorylation. The ability of insulin to induce the accumulation of Fra-1 mRNA, stimulate the fra-1 promoter and stimulate phosphorylation of Fra-1 all require the mitogen-activated protein (MAP) kinase cascade, which leads to the activation of extracellular-signal-regulated kinase (Erk) 1/2. Consequently, our results demonstrate that the Erk cascade plays a dual role in the co-ordinated regulation of the transcription and the phosphorylation of Fra-1 by insulin. PMID:12197835

  3. A maize mitogen-activated protein kinase kinase, ZmMKK1, positively regulated the salt and drought tolerance in transgenic Arabidopsis.

    PubMed

    Cai, Guohua; Wang, Guodong; Wang, Li; Liu, Yang; Pan, Jiaowen; Li, Dequan

    2014-07-15

    Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction modules in animals, plants and yeast. MAPK cascades are complicated networks and play vital roles in signal transduction pathways involved in biotic and abiotic stresses. In this study, a maize MAPKK gene, ZmMKK1, was characterized. Quantitative real time PCR (qRT-PCR) analysis demonstrated that ZmMKK1 transcripts were induced by diverse stresses and ABA signal molecule in maize root. Further study showed that the ZmMKK1-overexpressing Arabidopsis enhanced the tolerance to salt and drought stresses. However, seed germination, post-germination growth and stomatal aperture analysis demonstrated that ZmMKK1 overexpression was sensitive to ABA in transgenic Arabidopsis. Molecular genetic analysis revealed that the overexpression of ZmMKK1 in Arabidopsis enhanced the expression of ROS scavenging enzyme- and ABA-related genes, such as POD, CAT, RAB18 and RD29A under salt and drought conditions. In addition, heterologous overexpression of ZmMKK1 in yeast (Saccharomyces cerevisiae) improved the tolerance to salt and drought stresses. These results suggested that ZmMKK1 might act as an ABA- and ROS-dependent protein kinase in positive modulation of salt and drought tolerance. Most importantly, ZmMKK1 interacted with ZmMEKK1 as evidenced by yeast two-hybrid assay, redeeming a deficiency of MAPK interaction partners in maize. PMID:24974327

  4. Mitogen-activated protein kinase 4-like carrying an MEY motif instead of a TXY motif is involved in ozone tolerance and regulation of stomatal closure in tobacco.

    PubMed

    Yanagawa, Yuki; Yoda, Hiroshi; Osaki, Kohei; Amano, Yuta; Aono, Mitsuko; Seo, Shigemi; Kuchitsu, Kazuyuki; Mitsuhara, Ichiro

    2016-05-01

    The mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKK(EE)), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation. PMID:27126796

  5. Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

    PubMed Central

    Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

    2015-01-01

    Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5–MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases. PMID:26136265

  6. Nanoparticles up-regulate tumor necrosis factor-{alpha} and CXCL8 via reactive oxygen species and mitogen-activated protein kinase activation

    SciTech Connect

    Lee, Hye-Mi; Shin, D.-M.; Song, Hwan-Moon; Yuk, Jae-Min; Lee, Zee-Won; Lee, Sang-Hee; Hwang, Song Mei; Kim, Jin-Man; Lee, Chang-Soo Jo, Eun-Kyeong

    2009-07-15

    Evaluating the toxicity of nanoparticles is an integral aspect of basic and applied sciences, because imaging applications using traditional organic fluorophores are limited by properties such as photobleaching, spectral overlaps, and operational difficulties. This study investigated the toxicity of nanoparticles and their biological mechanisms. We found that nanoparticles, quantum dots (QDs), considerably activated the production of tumor necrosis factor (TNF)-{alpha} and CXC-chemokine ligand (CXCL) 8 through reactive oxygen species (ROS)- and mitogen-activated protein kinases (MAPKs)-dependent mechanisms in human primary monocytes. Nanoparticles elicited a robust activation of intracellular ROS, phosphorylation of p47phox, and nicotinamide adenine dinucleotide phosphate oxidase activities. Blockade of ROS generation with antioxidants significantly abrogated the QD-mediated TNF-{alpha} and CXCL8 expression in monocytes. The induced ROS generation subsequently led to the activation of MAPKs, which were crucial for mRNA and protein expression of TNF-{alpha} and CXCL8. Furthermore, confocal and electron microscopy analyses showed that internalized QDs were trapped in cytoplasmic vesicles and compartmentalized inside lysosomes. Finally, several repeated intravenous injections of QDs caused an increase in neutrophil infiltration in the lung tissues in vivo. These results provide novel insights into the QD-mediated chemokine induction and inflammatory toxic responses in vitro and in vivo.

  7. Molecular characterization of RsMPK2, a C1 subgroup mitogen-activated protein kinase in the desert plant Reaumuria soongorica.

    PubMed

    Liu, Yubing; Li, Xinrong; Tan, Huijuan; Liu, Meiling; Zhao, Xin; Wang, Jin

    2010-01-01

    Reaumuria soongorica (Pall.) Maxim. is a short woody shrub widely found in semi-arid areas of China, and can survive severe environmental stresses. To understand its potential signaling transduction pathway in stress tolerance, we investigated the participation of mitogen-activated protein kinases (MAPKs) as possible mediators of abiotic stresses. A novel MAP kinase cDNA (RsMPK2) that encodes a 374 amino acid protein was isolated from R. soongorica. RsMPK2 belongs to the C1 subgroup, which is still functionally uncharacterized compared to groups A and B; and contains all 11 of the conserved MAPK subdomains and the TEY phosphorylation motif. RsMPK2 is expressed in vegetative (root, stem, leaf and callus) and reproductive (flower) organs. The transcripts of RsMPK2 were rapidly accumulated at high levels when R. soongorica was subjected to dehydration, salinity conditions and treatment with abscisic acid or hydrogen peroxide. Growth analysis of Escherichia coli (srl::Tn10) cells transformed with pPROEXHT-RsMPK2 showed that the expression products of RsMPK2 do not act as an osmoprotectant. But, the inhibition of RsMPK2 expression by the inhibitor U0126 induced a decrease of antioxidant enzyme activity under stresses, indicating that RsMPK2 is involved in the regulation of the antioxidant defense system in the response to stress signaling. PMID:20833058

  8. Fast Retrograde Signaling in Response to High Light Involves Metabolite Export, MITOGEN-ACTIVATED PROTEIN KINASE6, and AP2/ERF Transcription Factors in Arabidopsis[C][W

    PubMed Central

    Vogel, Marc Oliver; Moore, Marten; König, Katharina; Pecher, Pascal; Alsharafa, Khalid; Lee, Justin; Dietz, Karl-Josef

    2014-01-01

    Regulation of the expression of nuclear genes encoding chloroplast proteins allows for metabolic adjustment in response to changing environmental conditions. This regulation is linked to retrograde signals that transmit information on the metabolic state of the chloroplast to the nucleus. Transcripts of several APETALA2/ETHYLENE RESPONSE FACTOR transcription factors (AP2/ERF-TFs) were found to respond within 10 min after transfer of low-light-acclimated Arabidopsis thaliana plants to high light. Initiation of this transcriptional response was completed within 1 min after transfer to high light. The fast responses of four AP2/ERF genes, ERF6, RRTF1, ERF104, and ERF105, were entirely deregulated in triose phosphate/phosphate translocator (tpt) mutants. Similarly, activation of MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) was upregulated after 1 min in the wild type but not in the tpt mutant. Based on this, together with altered transcript regulation in mpk6 and erf6 mutants, a retrograde signal transmission model is proposed starting with metabolite export through the triose phosphate/phosphate translocator with subsequent MPK6 activation leading to initiation of AP2/ERF-TF gene expression and other downstream gene targets. The results show that operational retrograde signaling in response to high light involves a metabolite-linked pathway in addition to previously described redox and hormonal pathways. PMID:24668746

  9. Melatonin alleviates myosin light chain kinase expression and activity via the mitogen-activated protein kinase pathway during atherosclerosis in rabbits.

    PubMed

    Cheng, Xiaowen; Wan, Yufeng; Xu, Yuanhong; Zhou, Qing; Wang, Yuan; Zhu, Huaqing

    2015-01-01

    Melatonin (MLT) is an endogenous indole compound with numerous biological activities that has been associated with atherosclerosis (AS). In the present study, rabbits were used as an AS model in order to investigate whether MLT affects endothelial cell permeability, myosin light chain kinase (MLCK) activity and MLCK expression via the mitogen-activated protein kinase (MAPK) pathway. Expression and activity of MLCK were measured using western blot analysis, quantitative polymerase chain reaction, immunohistochemistry and γ-32P-adenosine triphosphate incorporation. Endothelial permeability was detected using rhodamine phalloidin fluorescence staining. The phosphorylation of extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 in endothelial cells were also analyzed using western blot analysis. Atheromatous plaques were formed in rabbits with a high cholesterol diet; however, following treatment with MLT, the number and areas of atheromatous plaques were significantly reduced. In addition, MLT treatment reversed the increase of MLCK activity and expression that occurred in rabbits with high cholesterol intake. Furthermore, levels of phosphorylated ERK, JNK and p38 decreased following MLT treatment. In conclusion, the results of the present study indicated that AS may be associated with increased MLCK expression and activity, which was reduced following treatment with MLT. The mechanism of action of MLT was thought to proceed via modulating MAPK pathway signal transduction; however, further studies are required in order to fully elucidate the exact regulatory mechanisms involved. PMID:25339116

  10. Mitogen-activated protein kinase 4-like carrying an MEY motif instead of a TXY motif is involved in ozone tolerance and regulation of stomatal closure in tobacco

    PubMed Central

    Yanagawa, Yuki; Yoda, Hiroshi; Osaki, Kohei; Amano, Yuta; Aono, Mitsuko; Seo, Shigemi; Kuchitsu, Kazuyuki; Mitsuhara, Ichiro

    2016-01-01

    The mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKKEE), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation. PMID:27126796

  11. Counteractive Control of Polarized Morphogenesis during Mating by Mitogen-activated Protein Kinase Fus3 and G1 Cyclin-dependent Kinase

    PubMed Central

    Yu, Lu; Qi, Maosong; Sheff, Mark A.

    2008-01-01

    Cell polarization in response to external cues is critical to many eukaryotic cells. During pheromone-induced mating in Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) Fus3 induces polarization of the actin cytoskeleton toward a landmark generated by the pheromone receptor. Here, we analyze the role of Fus3 activation and cell cycle arrest in mating morphogenesis. The MAPK scaffold Ste5 is initially recruited to the plasma membrane in random patches that polarize before shmoo emergence. Polarized localization of Ste5 is important for shmooing. In fus3 mutants, Ste5 is recruited to significantly more of the plasma membrane, whereas recruitment of Bni1 formin, Cdc24 guanine exchange factor, and Ste20 p21-activated protein kinase are inhibited. In contrast, polarized recruitment still occurs in a far1 mutant that is also defective in G1 arrest. Remarkably, loss of Cln2 or Cdc28 cyclin-dependent kinase restores polarized localization of Bni1, Ste5, and Ste20 to a fus3 mutant. These and other findings suggest Fus3 induces polarized growth in G1 phase cells by down-regulating Ste5 recruitment and by inhibiting Cln/Cdc28 kinase, which prevents basal recruitment of Ste5, Cdc42-mediated asymmetry, and mating morphogenesis. PMID:18256288

  12. Regulation of p53, nuclear factor {kappa}B and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin

    SciTech Connect

    Kalra, Neetu; Bhui, Kulpreet; Roy, Preeti; Srivastava, Smita; George, Jasmine; Prasad, Sahdeo; Shukla, Yogeshwer

    2008-01-01

    Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-{kappa}B), we also investigated the effect of bromelain on Cox-2 and NF-{kappa}B expression. Results showed that bromelain application significantly inhibited Cox-2 and inactivated NF-{kappa}B by blocking phosphorylation and subsequent degradation of I{kappa}B{alpha}. In addition, bromelain treatment attenuated DMBA-TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that bromelain induces apoptosis-related proteins along with inhibition of NF-{kappa}B-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA-TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects.

  13. The Arabidopsis mitogen-activated protein kinase phosphatase PP2C5 affects seed germination, stomatal aperture, and abscisic acid-inducible gene expression.

    PubMed

    Brock, Anita K; Willmann, Roland; Kolb, Dagmar; Grefen, Laure; Lajunen, Heini M; Bethke, Gerit; Lee, Justin; Nürnberger, Thorsten; Gust, Andrea A

    2010-07-01

    Abscisic acid (ABA) is an important phytohormone regulating various cellular processes in plants, including stomatal opening and seed germination. Although protein phosphorylation via mitogen-activated protein kinases (MAPKs) has been suggested to be important in ABA signaling, the corresponding phosphatases are largely unknown. Here, we show that a member of the Protein Phosphatase 2C (PP2C) family in Arabidopsis (Arabidopsis thaliana), PP2C5, is acting as a MAPK phosphatase. The PP2C5 protein colocalizes and directly interacts with stress-induced MPK3, MPK4, and MPK6, predominantly in the nucleus. Importantly, altered PP2C5 levels affect MAPK activation. Whereas Arabidopsis plants depleted of PP2C5 show an enhanced ABA-induced activation of MPK3 and MPK6, ectopic expression of PP2C5 in tobacco (Nicotiana benthamiana) resulted in the opposite effect, with the two MAPKs salicylic acid-induced protein kinase and wound-induced protein kinase not being activated any longer after ABA treatment. Moreover, depletion of PP2C5, whose gene expression itself is affected by ABA treatment, resulted in altered ABA responses. Loss-of-function mutation in PP2C5 or AP2C1, a close PP2C5 homolog, resulted in an increased stomatal aperture under normal growth conditions and a partial ABA-insensitive phenotype in seed germination that was most prominent in the pp2c5 ap2c1 double mutant line. In addition, the response of ABA-inducible genes such as ABI1, ABI2, RD29A, and Erd10 was reduced in the mutant plants. Thus, we suggest that PP2C5 acts as a MAPK phosphatase that positively regulates seed germination, stomatal closure, and ABA-inducible gene expression. PMID:20488890

  14. Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress.

    PubMed

    Meili, Nicole; Christen, Verena; Fent, Karl

    2016-06-01

    Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1μM) and toxic concentrations (5μM) for 24, 48, and 72h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5μM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5nM, while HepG2 cells were insensitive up to 10μM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin. PMID:27061667

  15. The type 3 effector NopL of Sinorhizobium sp. strain NGR234 is a mitogen-activated protein kinase substrate.

    PubMed

    Ge, Ying-Ying; Xiang, Qi-Wang; Wagner, Christian; Zhang, Di; Xie, Zhi-Ping; Staehelin, Christian

    2016-04-01

    Pathogenic bacteria utilize type 3 secretion systems to inject type 3 effectors (T3Es) into host cells, thereby subverting host defense reactions. Similarly, T3Es of symbiotic nitrogen-fixing rhizobia can affect nodule formation on roots of legumes. Previous work showed that NopL (nodulation outer protein L) of Sinorhizobium(Ensifer) sp. strain NGR234 is multiply phosphorylated in eukaryotic cells and that this T3E suppresses responses mediated by mitogen-activated protein (MAP) kinase signaling in yeast (mating pheromone signaling) and plant cells (expression of pathogenesis-related defense proteins). Here, we show that NopL is a MAP kinase substrate. Microscopic observations of fluorescent fusion proteins and bimolecular fluorescence complementation analysis in onion cells indicated that NopL is targeted to the nucleus and forms a complex with SIPK (salicylic acid-induced protein kinase), a MAP kinase of tobacco. In vitro experiments demonstrated that NopL is phosphorylatyed by SIPK. At least nine distinct spots were observed after two-dimensional gel electrophoresis, indicating that NopL can be hyperphosphorylated by MAP kinases. Senescence symptoms in nodules of beans (Phaseolus vulgaris cv. Tendergreen) were analyzed to determine the symbiotic effector activity of different NopL variants with serine to alanine substitutions at identified and predicted phosphorylation sites (serine-proline motif). NopL variants with six or eight serine to alanine substitutions were partially active, whereas NopL forms with 10 or 12 substituted serine residues were inactive. In conclusion, our findings provide evidence that NopL interacts with MAP kinases and reveals the importance of serine-proline motifs for effector activity during symbiosis. PMID:26931172

  16. Expression of peptide fragments from proADM and involvement of mitogen-activated protein kinase signaling pathways in pulmonary remodeling induced by high pulmonary blood flow.

    PubMed

    Li, Wei; Guo, Aili; Wang, Lijuan; Kong, Qingyu; Wang, Rong; Han, Li; Zhao, Cuifen

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive pulmonary arterial remodeling and right ventricular failure. Despite recent advances in pathophysiological mechanism exploration and new therapeutic approaches, PAH remains a challenging condition. In this study, we investigated the roles of the peptide fragments from proadrenomedullin (proADM) such as adrenomedullin (ADM), adrenotensin (ADT), and proadrenomedullin N-terminal 20 peptide (PAMP) during pulmonary remodeling caused by high pulmonary blood flow, and probed the possible involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways. Sixteen rat models of PAH were artificially established by surgically connecting the left common carotid artery to the external jugular vein. We subcutaneously injected an extracellular signal-regulated protein kinase (ERK1/2) inhibitor, PD98059, in eight rats, treated another eight rats with an equal volume of saline. Eight rats without connections served as the control group. We observed that mRNA expression levels of ADM, stress-activated protein kinase (SAPK), and ERK1/2 were significantly elevated in the shunted rats; furthermore, ERK1/2 levels were significantly inhibited by PD98059. Protein levels of ADM, PAMP, p-SAPK, and p-ERK1/2 were significantly higher ADT was lower, and p-p38 remained unchanged in the rat models compared with the controls. However, the protein expression of both ADM and p-ERK1/2 was significantly inhibited by PD98059. Our results suggest that levels of ADM, ADT, and PAMP respond to pulmonary remodeling, and that activation of the SAPK and ERK1/2 signaling pathways is involved in pulmonary hypertension and artery remodeling caused by high pulmonary blood flow. PMID:25990643

  17. Filarial Lymphatic Pathology Reflects Augmented Toll-Like Receptor-Mediated, Mitogen-Activated Protein Kinase-Mediated Proinflammatory Cytokine Production ▿ †

    PubMed Central

    Babu, Subash; Anuradha, R.; Kumar, N. Pavan; George, P. Jovvian; Kumaraswami, V.; Nutman, Thomas B.

    2011-01-01

    Lymphatic filariasis can be associated with the development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Toll-like receptors (TLRs) are thought to play a major role in the development of filarial pathology. To elucidate the role of TLRs in the development of lymphatic pathology, we examined cytokine responses to different Toll ligands in patients with chronic lymphatic pathology (CP), infected patients with subclinical pathology (INF), and uninfected, endemic-normal (EN) individuals. TLR2, -7, and -9 ligands induced significantly elevated production of Th1 and other proinflammatory cytokines in CP patients in comparison to both INF and EN patients. TLR adaptor expression was not significantly different among the groups; however, both TLR2 and TLR9 ligands induced significantly higher levels of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein (MAP) kinases (MAPK) as well as increased activation of NF-κB in CP individuals. Pharmacologic inhibition of both ERK1/2 and p38 MAP kinase pathways resulted in significantly diminished production of proinflammatory cytokines in CP individuals. Our data, therefore, strongly suggest an important role for TLR2- and TLR9-mediated proinflammatory cytokine induction and activation of both the MAPK and NF-κB pathways in the development of pathology in human lymphatic filariasis. PMID:21875961

  18. Dual-specificity Phosphatase 1 Deficiency Induces Endometrioid Adenocarcinoma Progression via Activation of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Pathway

    PubMed Central

    Yang, Yuan; Zhou, Jing-Yi; Zhao, Li-Jun; Gao, Bao-Rong; Wan, Xiao-Ping; Wang, Jian-Liu

    2016-01-01

    Background: Previously, we reported that dual-specificity phosphatase 1 (DUSP1) was differentially expressed in endometrioid adenocarcinoma (EEA). However, the role of DUSP1 in EEA progression and the relationship between DUSP1 and medroxyprogesterone (MPA) are still unclear. Methods: The expression of DUSP1 in EEA specimens was detected by immunohistochemical analysis. The effect of DUSP1 on cell proliferation was analyzed by Cell Counting Kit 8 and colony formation assay, and cell migration was analyzed by transwell assay. MPA-induced DUSP1 expression in EEA cells was measured by Western blot. Results: DUSP1 expression was deficient in advanced International Federation of Gynecology and Obstetrics stage, high-grade and myometrial invasive EEA. In EEA cell lines (Hec1A, Hec1B, RL952, and Ishikawa), the DUSP1 expression was substantially higher in Ishikawa cells than in other cell lines (P < 0.05). Knockdown of DUSP1 promoted Ishikawa cells proliferation, migration, and activation of mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/Erk) pathway. MPA-induced DUSP1 expression and inhibited MAPK/Erk pathway in Ishikawa cells. Conclusions: Our data suggest that DUSP1 deficiency promotes EEA progression via MAPK/Erk pathway, which may be reversed by MPA, suggesting that DUSP1 may serve as a potential therapeutic target for the treatment of EEA. PMID:27174322

  19. Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway.

    PubMed

    Mai, Jietang; Nanayakkara, Gayani; Lopez-Pastrana, Jahaira; Li, Xinyuan; Li, Ya-Feng; Wang, Xin; Song, Ai; Virtue, Anthony; Shao, Ying; Shan, Huimin; Liu, Fang; Autieri, Michael V; Kunapuli, Satya P; Iwakura, Yoichiro; Jiang, Xiaohua; Wang, Hong; Yang, Xiao-Feng

    2016-03-01

    Interleukin-17 (IL-17)-secreting T helper 17 cells were recently identified as a CD4(+) T helper subset and implicated in various inflammatory and autoimmune diseases. The issues of whether and by what mechanism hyperlipidemic stress induces IL-17A to activate aortic endothelial cells (ECs) and enhance monocyte adhesion remained largely unknown. Using biochemical, immunological, microarray, experimental data mining analysis, and pathological approaches focused on primary human and mouse aortic ECs (HAECs and MAECs) and our newly generated apolipoprotein E (ApoE)(-/-)/IL-17A(-/-) mice, we report the following new findings. 1) The hyperlipidemia stimulus oxidized low density lipoprotein up-regulated IL-17 receptor(s) in HAECs and MAECs. 2) IL-17A activated HAECs and increased human monocyte adhesion in vitro. 3) A deficiency of IL-17A reduced leukocyte adhesion to endothelium in vivo. 3) IL-17A activated HAECs and MAECs via up-regulation of proinflammatory cytokines IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine CXC motif ligand 1 (CXCL1), and CXCL2. 4) IL-17A activated ECs specifically via the p38 mitogen-activated protein kinases (MAPK) pathway; the inhibition of p38 MAPK in ECs attenuated IL-17A-mediated activation by ameliorating the expression of the aforementioned proinflammatory cytokines, chemokines, and EC adhesion molecules including intercellular adhesion molecule 1. Taken together, our results demonstrate for the first time that IL-17A activates aortic ECs specifically via p38 MAPK pathway. PMID:26733204

  20. Involvement of mitogen-activated protein kinases and NF{kappa}B in LPS-induced CD40 expression on human monocytic cells

    SciTech Connect

    Wu Weidong | Alexis, Neil E. |; Chen Xian |; Bromberg, Philip A. |; Peden, David B. ||

    2008-04-15

    CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NF{kappa}B were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NF{kappa}B activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NF{kappa}B activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NF{kappa}B activation, and CD40 expression. Moreover, blockage of MAPK and NF{kappa}B activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NF{kappa}B.

  1. Mitogen-Activated Protein Kinase Cascade MKK7-MPK6 Plays Important Roles in Plant Development and Regulates Shoot Branching by Phosphorylating PIN1 in Arabidopsis.

    PubMed

    Jia, Weiyan; Li, Baohua; Li, Shujia; Liang, Yan; Wu, Xiaowei; Ma, Mei; Wang, Jiyao; Gao, Jin; Cai, Yueyue; Zhang, Yuanya; Wang, Yingchun; Li, Jiayang; Wang, Yonghong

    2016-09-01

    Emerging evidences exhibit that mitogen-activated protein kinase (MAPK/MPK) signaling pathways are connected with many aspects of plant development. The complexity of MAPK cascades raises challenges not only to identify the MAPK module in planta but also to define the specific role of an individual module. So far, our knowledge of MAPK signaling has been largely restricted to a small subset of MAPK cascades. Our previous study has characterized an Arabidopsis bushy and dwarf1 (bud1) mutant, in which the MAP Kinase Kinase 7 (MKK7) was constitutively activated, resulting in multiple phenotypic alterations. In this study, we found that MPK3 and MPK6 are the substrates for phosphorylation by MKK7 in planta. Genetic analysis showed that MKK7-MPK6 cascade is specifically responsible for the regulation of shoot branching, hypocotyl gravitropism, filament elongation, and lateral root formation, while MKK7-MPK3 cascade is mainly involved in leaf morphology. We further demonstrated that the MKK7-MPK6 cascade controls shoot branching by phosphorylating Ser 337 on PIN1, which affects the basal localization of PIN1 in xylem parenchyma cells and polar auxin transport in the primary stem. Our results not only specify the functions of the MKK7-MPK6 cascade but also reveal a novel mechanism for PIN1 phosphorylation, establishing a molecular link between the MAPK cascade and auxin-regulated plant development. PMID:27618482

  2. SB203580, a p38 mitogen-activated protein kinase inhibitor, fails to improve functional outcome following a moderate spinal cord injury in rat.

    PubMed

    Stirling, D P; Liu, J; Plunet, W; Steeves, J D; Tetzlaff, W

    2008-07-31

    We examined the spatial and temporal expression patterns of active p38 mitogen-activated protein kinase (MAPK), an important regulator of immune cell function, following spinal cord injury (SCI). We further assessed whether administration of SB203580, an inhibitor of p38 MAPK activity, would reduce inflammation, improve tissue sparing, and improve functional outcome after SCI. Adult Wistar rats were subjected to a T9/10 SCI contusion of moderate severity and killed at several time points after injury, whereas sham-injured (control) animals only received a laminectomy. In control animals, active p38 MAPK expression was primarily localized to resting microglia within the spinal cord. Over the first 24 h after SCI, a continuing increase in active p38 MAPK expression was evident in neutrophils and activated microglia (OX42+) surrounding the spinal lesion site. At 15 days post-injury, active p38 MAPK was localized to macrophages (ED1+) that now dominated the lesion site. In addition, active p38 MAPK was localized to macrophages within white matter fiber tracts undergoing degeneration, several segments rostral and caudal to the injury site, which persisted for at least 6 weeks. Overall, our results demonstrate that active p38 MAPK is increased within resident and invading immune cells after SCI contusion injury and, therefore, may be an important target to regulate the inflammatory cascade after SCI. However, intrathecal application of SB203580 failed to improve functional outcome after a moderate SCI contusion. PMID:18562123

  3. Protective effect of paeoniflorin on irradiation-induced cell damage involved in modulation of reactive oxygen species and the mitogen-activated protein kinases.

    PubMed

    Li, Chun Rong; Zhou, Zhe; Zhu, Dan; Sun, Yu Ning; Dai, Jin Ming; Wang, Sheng Qi

    2007-01-01

    Ionizing radiation can induce DNA damage and cell death by generating reactive oxygen species (ROS). The objective of this study was to investigate the radioprotective effect of paeoniflorin (PF, a main bioactive component in the traditional Chinese herb peony) on irradiated thymocytes and discover the possible mechanisms of protection. We found 60Co gamma-ray irradiation increased cell death and DNA fragmentation in a dose-dependent manner while increasing intracellular ROS. Pretreatment of thymocytes with PF (50-200 microg/ml) reversed this tendency and attenuated irradiation-induced ROS generation. Hydroxyl-scavenging action of PF in vitro was detected through electron spin resonance assay. Several anti-apoptotic characteristics of PF, including the ability to diminish cytosolic Ca2+ concentration, inhibit caspase-3 activation, and upregulate Bcl-2 and downregulate Bax in 4Gy-irradiated thymocytes were determined. Extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 kinase were activated by 4Gy irradiation, whereas its activations were partly blocked by pretreatment of cells with PF. The presence of ERK inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580 decreased cell death in 4Gy-irradiated thymocytes. These results suggest PF protects thymocytes against irradiation-induced cell damage by scavenging ROS and attenuating the activation of the mitogen-activated protein kinases. PMID:17097910

  4. Epoxyeicosatrienoic acids prevent cisplatin-induced renal apoptosis through a p38 mitogen-activated protein kinase-regulated mitochondrial pathway.

    PubMed

    Liu, Yingmei; Lu, Xiaodan; Nguyen, Sinh; Olson, Jean L; Webb, Heather K; Kroetz, Deanna L

    2013-12-01

    Soluble epoxide hydrolase (sEH) catalyzes the conversion of epoxyeicosatrienoic acids into less active eicosanoids, and inhibitors of sEH have anti-inflammatory and antiapoptotic properties. Based on previous observations that sEH inhibition attenuates cisplatin-induced nephrotoxicity by modulating nuclear factor-κB signaling, we hypothesized that this strategy would also attenuate cisplatin-induced renal apoptosis. Inhibition of sEH with AR9273 [1-adamantan-1-yl-3-(1-methylsulfonyl-piperidin-4-yl-urea)] reduced cisplatin-induced apoptosis through mechanisms involving mitochondrial apoptotic pathways and by reducing reactive oxygen species. Renal mitochondrial Bax induction following cisplatin treatment was significantly decreased by treatment of mice with AR9273 and these antiapoptotic effects involved p38 mitogen-activated protein kinase signaling. Similar mechanisms contributed to reduced apoptosis in Ephx2(-/-) mice treated with cisplatin. Moreover, in pig kidney proximal tubule cells, cisplatin-induced mitochondrial trafficking of Bax and cytochrome c, caspase-3 activation, and oxidative stress are significantly attenuated in the presence of epoxyeicosatrienoic acids (EETs). Collectively, these in vivo and in vitro studies demonstrate a role for EETs in limiting cisplatin-induced renal apoptosis. Inhibition of sEH represents a novel therapeutic strategy for protection against cisplatin-induced renal damage. PMID:24092818

  5. Epoxyeicosatrienoic Acids Prevent Cisplatin-Induced Renal Apoptosis through a p38 Mitogen-Activated Protein Kinase–Regulated Mitochondrial Pathway

    PubMed Central

    Liu, Yingmei; Lu, Xiaodan; Nguyen, Sinh; Olson, Jean L.; Webb, Heather K.

    2013-01-01

    Soluble epoxide hydrolase (sEH) catalyzes the conversion of epoxyeicosatrienoic acids into less active eicosanoids, and inhibitors of sEH have anti-inflammatory and antiapoptotic properties. Based on previous observations that sEH inhibition attenuates cisplatin-induced nephrotoxicity by modulating nuclear factor-κB signaling, we hypothesized that this strategy would also attenuate cisplatin-induced renal apoptosis. Inhibition of sEH with AR9273 [1-adamantan-1-yl-3-(1-methylsulfonyl-piperidin-4-yl-urea)] reduced cisplatin-induced apoptosis through mechanisms involving mitochondrial apoptotic pathways and by reducing reactive oxygen species. Renal mitochondrial Bax induction following cisplatin treatment was significantly decreased by treatment of mice with AR9273 and these antiapoptotic effects involved p38 mitogen-activated protein kinase signaling. Similar mechanisms contributed to reduced apoptosis in Ephx2−/− mice treated with cisplatin. Moreover, in pig kidney proximal tubule cells, cisplatin-induced mitochondrial trafficking of Bax and cytochrome c, caspase-3 activation, and oxidative stress are significantly attenuated in the presence of epoxyeicosatrienoic acids (EETs). Collectively, these in vivo and in vitro studies demonstrate a role for EETs in limiting cisplatin-induced renal apoptosis. Inhibition of sEH represents a novel therapeutic strategy for protection against cisplatin-induced renal damage. PMID:24092818

  6. Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

    PubMed Central

    Lu, Qing; Rau, Thomas F.; Harris, Valerie; Johnson, Maribeth; Poulsen, David J.; Black, Stephen M.

    2016-01-01

    The pathological basis of neonatal hypoxia–ischemia (HI) brain damage is characterized by neuronal cell loss. Oxidative stress is thought to be one of the main causes of HI-induced neuronal cell death. The p38 mitogen-activated protein kinase (MAPK) is activated under conditions of cell stress. However, its pathogenic role in regulating the oxidative stress associated with HI injury in the brain is not well understood. Thus, this study was conducted to examine the role of p38 MAPK signaling in neonatal HI brain injury using neonatal rat hippocampal slice cultures exposed to oxygen / glucose deprivation (OGD). Our results indicate that OGD led to a transient increase in p38 MAPK activation that preceded increases in superoxide generation and neuronal death. This increase in neuronal cell death correlated with an increase in the activation of caspase-3 and the appearance of apoptotic neuronal cells. Pre-treatment of slice cultures with the p38 MAPK inhibitor, SB203580, or the expression of an antisense p38 MAPK construct only in neuronal cells, through a Synapsin I-1-driven adeno-associated virus vector, inhibited p38 MAPK activity and exerted a neuroprotective effect as demonstrated by decreases in OGD-mediated oxidative stress, caspase activation and neuronal cell death. Thus, we conclude that the activation of p38 MAPK in neuronal cells plays a key role in the oxidative stress and neuronal cell death associated with OGD. PMID:21939459

  7. Interleukin 17A promotes pneumococcal clearance by recruiting neutrophils and inducing apoptosis through a p38 mitogen-activated protein kinase-dependent mechanism in acute otitis media.

    PubMed

    Wang, Wei; Zhou, Aie; Zhang, Xuemei; Xiang, Yun; Huang, Yifei; Wang, Lei; Zhang, Shuai; Liu, Yusi; Yin, Yibing; He, Yujuan

    2014-06-01

    Streptococcus pneumoniae is a Gram-positive and human-restricted pathogen colonizing the nasopharynx with an absence of clinical symptoms as well as a major pathogen causing otitis media (OM), one of the most common childhood infections. Upon bacterial infection, neutrophils are rapidly activated and recruited to the infected site, acting as the frontline defender against emerging microbial pathogens via different ways. Evidence shows that interleukin 17A (IL-17A), a neutrophil-inducing factor, plays important roles in the immune responses in several diseases. However, its function in response to S. pneumoniae OM remains unclear. In this study, the function of IL-17A in response to S. pneumoniae OM was examined using an in vivo model. We developed a model of acute OM (AOM) in C57BL/6 mice and found that neutrophils were the dominant immune cells that infiltrated to the middle ear cavity (MEC) and contributed to bacterial clearance. Using IL-17A knockout (KO) mice, we found that IL-17A boosted neutrophil recruitment to the MEC and afterwards induced apoptosis, which was identified to be conducive to bacterial clearance. In addition, our observation suggested that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was involved in the recruitment and apoptosis of neutrophils mediated by IL-17A. These data support the conclusion that IL-17A contributes to the host immune response against S. pneumoniae by promoting neutrophil recruitment and apoptosis through the p38 MAPK signaling pathway. PMID:24664502

  8. Inhibition of p38 Mitogen-Activated Protein Kinase Enhances the Apoptosis Induced by Oxidized Low-Density Lipoprotein in Endothelial Progenitor Cells.

    PubMed

    Tie, Guodong; Yan, Jinglian; Messina, Julia A; Raffai, Robert L; Messina, Louis M

    2015-01-01

    Oxidized low-density lipoprotein (oxLDL) is an important risk factor in the development of atherosclerosis. oxLDL has been shown to decrease endothelial progenitor cell (EPC) number by inducing apoptosis. p38 mitogen-activated protein kinase (MAPK) was shown to be activated by oxLDL and participated in the regulation of EPC number and function. However, the role of p38 remains unknown. Here, we show that oxLDL-induced p38 phosphorylation in EPCs is time and dose dependent. Treatment with antioxidant N-acetyl cysteine restored oxLDL-induced p38 phosphorylation to basal levels. LOX-1-blocking antibody also significantly decreased oxLDL-induced p38 phosphorylation. Interestingly, TUNEL staining showed that pretreatment with the p38 inhibitor SB203580 further increased oxLDL-induced apoptosis in EPCs. In accordance with these findings, pretreatment with SB203580 further attenuated Akt phosphorylation in EPCs challenged with oxLDL, indicating an interaction between Akt and p38 MAPK pathways. In agreement, inhibition of p38 MAPK further attenuated Akt phosphorylation and increased apoptosis in EPCs isolated from hypercholesterolemic ApoE-/- mice. In conclusion, p38 MAPK serves as an anti-apoptotic pathway by supporting Akt activity when EPCs are challenged with oxLDL. PMID:27031525

  9. Anthrax lethal toxin impairs CD1d-mediated antigen presentation by targeting the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway.

    PubMed

    Khan, Masood A; Gallo, Richard M; Brutkiewicz, Randy R

    2010-05-01

    Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis and an important means by which this bacterium evades the host's immune system. In this study, we demonstrate that CD1d-expressing cells treated with LT have reduced CD1d-mediated antigen presentation. We earlier showed an important role for the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulation of CD1d-mediated antigen presentation, and we report here that LT impairs antigen presentation by CD1d in an ERK1/2-dependent manner. Similarly, LT and the ERK1/2 pathway-specific inhibitor U0126 caused a decrease in major histocompatibility complex (MHC) class II-mediated antigen presentation. Confocal microscopy analyses revealed altered intracellular distribution of CD1d and LAMP-1 in LT-treated cells, similar to the case for ERK1/2-inhibited cells. These results suggest that Bacillus anthracis has the ability to evade the host's innate immune system by reducing CD1d-mediated antigen presentation through targeting the ERK1/2 pathway. PMID:20194602

  10. Violacein induces p44/42 mitogen-activated protein kinase-mediated solid tumor cell death and inhibits tumor cell migration

    PubMed Central

    MEHTA, TORAL; VERCRUYSSE, KOEN; JOHNSON, TERRANCE; EJIOFOR, ANTHONY OKECHUKWU; MYLES, ELBERT; QUICK, QUINCY ANTOINE

    2015-01-01

    Microbial secondary metabolites have emerged as alternative novel drugs for the treatment of human cancers. Violacein, a purple pigment produced by Chromobacterium violaceum, was investigated in the present study for its anti-tumor properties in tumor cell lines. Clinically applicable concentrations of violacein were demonstrated to inhibit the proliferative capacity of tumor cell lines according to a crystal violet proliferation assay. The underlying mechanism was the promotion of apoptotic cell death, as indicated by poly(ADP ribose) polymerase cleavage and p44/42 mitogen-activated protein kinase signaling determined by western blot analysis. Collectively, this provided mechanistic evidence that violacein elicits extracellular-signal regulated kinase-induced apoptosis via the intrinsic pathway. The anti-malignant properties of violacein in the present study were further demonstrated by its inhibitory effects on brain tumor cell migration, specifically glioblastomas, one of the most invasive and therapeutically resistant neoplasms in the clinic. Additionally, solid tumors examined in the present study displayed differential cellular responses and sensitivities to violacein as observed by morphologically induced cellular changes that contributed to its anti-migratory properties. In conclusion, violacein is a novel natural product with the potential to kill several types of human tumor cell lines, as well as prevent disease recurrence by antagonizing cellular processes that contribute to metastatic invasion. PMID:25816226

  11. Isoflurane attenuates mouse microglial engulfment induced by lipopolysaccharide and interferon-γ possibly by inhibition of p38 mitogen-activated protein kinase.

    PubMed

    Ryu, Jung-Hee; Wang, Zhi; Fan, Dan; Han, Sung-Hee; Do, Sang-Hwan; Zuo, Zhiyi

    2016-09-28

    Microglial engulfment is a basic function to clean up dead and injured cells and invaders, such as bacteria. This study was designed to assess the effects of isoflurane on the microglial engulfment induced by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) and the involvement of p38 mitogen-activated protein kinase (MAPK) in these effects. C8-B4 microglial cells were exposed to 1, 2, and 3% isoflurane at 2 h after the initiation of LPS (100 ng/ml) and IFN-γ (1 ng/ml) stimulation. Fluorescent immunostaining was performed to assess the percentage of cells with engulfment of fluorescent microspheres after stimulation for 24 h. P38 and phosphorylated p38 were determined by Western blotting. Isoflurane concentration dependently decreased microglial engulfment stimulated by LPS and IFN-γ. LPS and IFN-γ increased the phosphorylated p38 in microglial cells. This upregulation was decreased by isoflurane. SB203580, a p38 MAPK inhibitor, abolished the LPS-induced and IFN-γ-induced increase of engulfment activity, whereas anisomycin, a p38 MAPK activator, partly reversed the isoflurane-decreased microglial engulfment activity. These results suggest that isoflurane reduces LPS-induced and IFN-γ-induced microglial engulfment and that these effects may be mediated by inhibiting p38 MAPK. PMID:27513199

  12. The mitogen-activated protein kinase gene, VdHog1, regulates osmotic stress response, microsclerotia formation and virulence in Verticillium dahliae.

    PubMed

    Wang, Yonglin; Tian, Longyan; Xiong, Dianguang; Klosterman, Steven J; Xiao, Shuxiao; Tian, Chengming

    2016-03-01

    The fungus Verticillium dahliae has gained worldwide notoriety as a destructive plant pathogen, causing vascular wilt diseases on diverse plant species. V. dahliae produces melanized resting bodies, known as microsclerotia, which can survive for 15years in the soil, and are thus critically important in its disease cycle. However, the molecular mechanisms that underpin microsclerotia formation, survival, and germination remain poorly understood. In this study, we observed that deletion of VdHog1 (ΔVdHog1), encoding a homolog of a high-osmolarity glycerol (HOG) response mitogen-activated protein kinase, displayed decreased numbers of melanized microsclerotia in culture, heightened sensitivity to hyperosmotic stress, and increased resistance to the fungicide fludioxonil. Through RNA-Seq analysis, we identified 221 genes differentially expressed in the ΔVdHog1 strain. Interestingly, the expression levels of genes involved in melanin biosynthesis, as well as the hydrophobin gene VDH1, involved in the early stage of microsclerotia formation, were significantly decreased in the ΔVdHog1 strains relative to the wild-type expression levels. The ΔVdHog1 strains exhibited decreased virulence relative to the wild type strain on smoke tree seedlings. These results indicate that VdHog1 regulates hyperosmotic stress responses in V. dahliae, and establishes the Hog1-mediated pathway as a target to further probe the up- and downstream processes that regulate asexual development in this fungus. PMID:26812120

  13. Docetaxel enhances apoptosis and G2/M cell cycle arrest by suppressing mitogen-activated protein kinase signaling in human renal clear cell carcinoma.

    PubMed

    Han, T D; Shang, D H; Tian, Y

    2016-01-01

    Tremendous efforts have been made in renal cell carcinoma (RCC) patients' research; however, clinical findings in patients have been disappointing. The aims of our study were to identify better or alternative therapeutic methods that can reverse chemotherapy resistance and to enhance sensitivity to docetaxel (DOX)-based chemotherapy drugs. We evaluated the anti-proliferative effect of DOX against RCC cells. DOX was found to suppress proliferation of RCC cells under in vitro and in vivo settings. Flow cytometric analysis revealed that DOX suppressed cell growth by induction of both apoptosis and G2/M cell cycle arrest in a dose-dependent manner. Various patterns of gene expression were observed by cluster analysis. In addition, based on network analysis using the ingenuity pathway analysis software, DOX was found to suppress phosphorylation of extracellular signal-regulated kinase 1/2 and p38, suggesting that the mitogen-activated protein kinase signaling pathway plays a vital role in the anti-proliferative effect of DOX against RCC. PMID:26909952

  14. Silencing of WIPK and SIPK mitogen-activated protein kinases reduces tobacco mosaic virus accumulation but permits systemic viral movement in tobacco possessing the N resistance gene.

    PubMed

    Kobayashi, Michie; Seo, Shigemi; Hirai, Katsuyuki; Yamamoto-Katou, Ayako; Katou, Shinpei; Seto, Hideharu; Meshi, Tetsuo; Mitsuhara, Ichiro; Ohashi, Yuko

    2010-08-01

    Infection of tobacco cultivars possessing the N resistance gene with Tobacco mosaic virus (TMV) results in confinement of the virus by necrotic lesions at the infection site. Although the mitogen-activated protein kinases WIPK and SIPK have been implicated in TMV resistance, evidence linking them directly to disease resistance is, as yet, insufficient. Viral multiplication was reduced slightly in WIPK- or SIPK-silenced plants but substantially in WIPK/SIPK-silenced plants, and was correlated with an increase in salicylic acid (SA) and a decrease in jasmonic acid (JA). Silencing of WIPK and SIPK in a tobacco cultivar lacking the N gene did not inhibit viral accumulation. The reduction in viral accumulation was attenuated by expressing a gene for an SA-degrading enzyme or by exogenously applying JA. Inoculation of lower leaves resulted in the systemic spread of TMV and formation of necrotic lesions in uninoculated upper leaves. These results suggested that WIPK and SIPK function to negatively regulate local resistance to TMV accumulation, partially through modulating accumulation of SA and JA in an N-dependent manner, but positively regulate systemic resistance. PMID:20615114

  15. The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development

    PubMed Central

    Lafleur, Edith; Kapfer, Christelle; Joly, Valentin; Liu, Yang; Tebbji, Faiza; Daigle, Caroline; Gray-Mitsumune, Madoka; Cappadocia, Mario; Nantel, André; Matton, Daniel P.

    2015-01-01

    The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development. PMID:25576576

  16. Genome-Wide Identification of Mitogen-Activated Protein Kinase Gene Family across Fungal Lineage Shows Presence of Novel and Diverse Activation Loop Motifs

    PubMed Central

    Mohanta, Tapan Kumar; Mohanta, Nibedita; Parida, Pratap; Panda, Sujogya Kumar; Ponpandian, Lakshmi Narayanan; Bae, Hanhong

    2016-01-01

    The mitogen-activated protein kinase (MAPK) is characterized by the presence of the T-E-Y, T-D-Y, and T-G-Y motifs in its activation loop region and plays a significant role in regulating diverse cellular responses in eukaryotic organisms. Availability of large-scale genome data in the fungal kingdom encouraged us to identify and analyse the fungal MAPK gene family consisting of 173 fungal species. The analysis of the MAPK gene family resulted in the discovery of several novel activation loop motifs (T-T-Y, T-I-Y, T-N-Y, T-H-Y, T-S-Y, K-G-Y, T-Q-Y, S-E-Y and S-D-Y) in fungal MAPKs. The phylogenetic analysis suggests that fungal MAPKs are non-polymorphic, had evolved from their common ancestors around 1500 million years ago, and are distantly related to plant MAPKs. We are the first to report the presence of nine novel activation loop motifs in fungal MAPKs. The specificity of the activation loop motif plays a significant role in controlling different growth and stress related pathways in fungi. Hence, the presences of these nine novel activation loop motifs in fungi are of special interest. PMID:26918378

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

    PubMed Central

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

    2015-01-01

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

  18. The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

    PubMed Central

    Choi, T; Fukasawa, K; Zhou, R; Tessarollo, L; Borror, K; Resau, J; Vande Woude, G F

    1996-01-01

    Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692939

  19. Lactobacillus acidophilus induces cytokine and chemokine production via NF-κB and p38 mitogen-activated protein kinase signaling pathways in intestinal epithelial cells.

    PubMed

    Jiang, Yujun; Lü, Xuena; Man, Chaoxin; Han, Linlin; Shan, Yi; Qu, Xingguang; Liu, Ying; Yang, Shiqin; Xue, Yuqing; Zhang, Yinghua

    2012-04-01

    Intestinal epithelial cells can respond to certain bacteria by producing an array of cytokines and chemokines which are associated with host immune responses. Lactobacillus acidophilus NCFM is a characterized probiotic, originally isolated from human feces. This study aimed to test the ability of L. acidophilus NCFM to stimulate cytokine and chemokine production in intestinal epithelial cells and to elucidate the mechanisms involved in their upregulation. In experiments using intestinal epithelial cell lines and mouse models, we observed that L. acidophilus NCFM could rapidly but transiently upregulate a number of effector genes encoding cytokines and chemokines such as interleukin 1α (IL-1α), IL-1β, CCL2, and CCL20 and that cytokines showed lower expression levels with L. acidophilus NCFM treatment than chemokines. Moreover, L. acidophilus NCFM could activate a pathogen-associated molecular pattern receptor, Toll-like receptor 2 (TLR2), in intestinal epithelial cell lines. The phosphorylation of NF-κB p65 and p38 mitogen-activated protein kinase (MAPK) in intestinal epithelial cell lines was also enhanced by L. acidophilus NCFM. Furthermore, inhibitors of NF-κB (pyrrolidine dithiocarbamate [PDTC]) and p38 MAPK (SB203580) significantly reduced cytokine and chemokine production in the intestinal epithelial cell lines stimulated by L. acidophilus NCFM, suggesting that both NF-κB and p38 MAPK signaling pathways were important for the production of cytokines and chemokines induced by L. acidophilus NCFM. PMID:22357649

  20. Zinc oxide influences mitogen-activated protein kinase and TGF-β1 signaling pathways, and enhances intestinal barrier integrity in weaned pigs.

    PubMed

    Song, Ze He; Xiao, Kan; Ke, Ya Lu; Jiao, Le Fei; Hu, Cai Hong

    2015-05-01

    Weaning is the most significant event in the life of pigs and is always related with intestinal disruption. Although it is well known that zinc oxide (ZnO) exerts beneficial effects on the intestinal barrier, the mechanisms underlying these effects have not yet been fully elucidated. We examined whether ZnO protects the intestinal barrier via mitogen-activated protein kinases and TGF-β1 signaling pathways. Twelve barrows weaned at 21 d of age were randomly assigned to two treatments (0 verus 2200 mg Zn/kg from ZnO) for 1 wk. The results showed that supplementation with ZnO increased daily gain and feed intake, and decreased postweaning scour scores. ZnO improved intestinal morphology, as indicated by increased villus height and villus height:crypt depth ratio, and intestinal barrier function, indicated by increased transepithelial electrical resistance and decreased mucosal-to-serosal permeability to 4-ku FITC dextran. ZnO decreased the ratios of the phosphorylated to total JNK and p38 (p-JNK/JNK and p-p38/p38), while it increased the ratio of ERK (p-ERK/ERK). Supplementation with ZnO increased intestinal TGF-β1 expression. The results indicate that supplementation with ZnO activates ERK ½, and inhibits JNK and p38 signaling pathways, and increases intestinal TGF-β1 expression in weaned pigs. PMID:24917655

  1. CpG oligodeoxynucleotides induce IL-8 expression in CD34+ cells via mitogen-activated protein kinase-dependent and NF-kappaB-independent pathways.

    PubMed

    Kim, Jung Mogg; Kim, Nam In; Oh, Yu-Kyoung; Kim, Young-Jeon; Youn, Jeehee; Ahn, Myung-Ju

    2005-12-01

    To elucidate the role of Toll-like receptor 9 (TLR9) activation along with the intracellular signaling pathways triggered by CpG DNA in CD34+ cells, we investigated whether synthetic oligodeoxynucleotides (ODNs), containing unmethylated CpG motifs, could induce IL-8 expression in CD34+ cells through mitogen-activated protein kinase (MAPK) or nuclear factor-kappaB (NF-kappaB) pathway. We demonstrated evidence for the first time that CD34+ cells constitutively expressed TLR9. Exposure of the cells to CpG ODN resulted in a time- and dose-dependent increase of IL-8 expression, and activation of phosphorylated ERK1/2 and phosphorylated p38. In addition, CpG ODN stimulated AP-1, but not NF-kappaB, signals. Moreover, inhibitors of MAPK (U0126 and SB203580) significantly reduced the IL-8 production, while the inhibition of NF-kappaB (pyrrolidinedithiocarbamate and retrovirus containing dominant-negative IkappaB alpha plasmid) did not affect the IL-8 expression increased by CpG ODN. Moreover, co-stimulation with LPS and CpG synergistically up-regulates IL-8 in CD34+ cells. These results suggest that CpG DNA, acting on TLR9, activates CD34+ cells to express IL-8 through MAPK-dependent and NF-kappaB-independent pathways. PMID:16263754

  2. The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development.

    PubMed

    Lafleur, Edith; Kapfer, Christelle; Joly, Valentin; Liu, Yang; Tebbji, Faiza; Daigle, Caroline; Gray-Mitsumune, Madoka; Cappadocia, Mario; Nantel, André; Matton, Daniel P

    2015-04-01

    The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development. PMID:25576576

  3. Enediyne lidamycin induces apoptosis in human multiple myeloma cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase.

    PubMed

    Zhen, Yong-Zhan; Lin, Ya-Jun; Shang, Bo-Yang; Zhen, Yong-Su

    2009-07-01

    In the present study, the effects of lidamycin (LDM), a member of the enediyne antibiotic family, on two human multiple myeloma (MM) cell lines, U266 and SKO-007, were evaluated. In MTS assay, LDM showed much more potent cytotoxicity than conventional anti-MM agents to both cell lines. The IC(50) values of LDM for the U266 and SKO-007 cells were 0.0575 +/- 0.0015 and 0.1585 +/- 0.0166 nM, respectively, much lower than those of adriamycin, dexamethasone, and vincristine. Mechanistically, LDM triggered MM cells apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in LDM-induced cell death. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed the LDM-induced apoptosis through decreasing the level of cleaved PARP and caspase-3/7. Interestingly, phosphorylation of extracellular signal-related kinase was increased by LDM; conversely, MEK inhibitor synergistically enhanced LDM-induced cytotoxicity and apoptosis in MM cells. The results demonstrated that LDM suppresses MM cell growth through the activation of p38 MAPK and JNK, with the potential to be developed as a chemotherapeutic agent for MM. PMID:19468799

  4. Cloning and molecular characterization of a mitogen-activated protein kinase gene from Poncirus trifoliata whose ectopic expression confers dehydration/drought tolerance in transgenic tobacco

    PubMed Central

    Huang, Xiao-San; Luo, Tao; Fu, Xing-Zheng; Fan, Qi-Jun; Liu, Ji-Hong

    2011-01-01

    The mitogen-activated protein kinase (MAPK) cascade plays pivotal roles in diverse signalling pathways related to plant development and stress responses. In this study, the cloning and functional characterization of a group-I MAPK gene, PtrMAPK, in Poncirus trifoliata (L.) Raf are reported. PtrMAPK contains 11 highly conserved kinase domains and a phosphorylation motif (TEY), and is localized in the nucleus of transformed onion epidermal cells. The PtrMAPK transcript level was increased by dehydration and cold, but was unaffected by salt. Transgenic overexpression of PtrMAPK in tobacco confers dehydration and drought tolerance. The transgenic plants exhibited better water status, less reactive oxygen species (ROS) generation, and higher levels of antioxidant enzyme activity and metabolites than the wild type. Interestingly, the stress tolerance capacity of the transgenic plants was compromised by inhibitors of antioxidant enzymes. In addition, overexpression of PtrMAPK enhanced the expression of ROS-related and stress-responsive genes under normal or drought conditions. Taken together, these data demonstrate that PtrMAPK acts as a positive regulator in dehydration/drought stress responses by either regulating ROS homeostasis through activation of the cellular antioxidant systems or modulating transcriptional levels of a variety of stress-associated genes. PMID:21778184

  5. Disruption of SRM1, a mitogen-activated protein kinase gene, affects sensitivity to osmotic and ultraviolet stressors in the phytopathogenic fungus Bipolaris oryzae.

    PubMed

    Moriwaki, Akihiro; Kubo, Emiko; Arase, Sakae; Kihara, Junichi

    2006-04-01

    Mitogen-activated protein kinases (MAPKs) play key roles in biological processes including differentiation, growth, proliferation, survival, and stress responses. We isolated and characterized the SRM1 gene, which encodes an MAPK related to yeast High-osmolarity glycerol 1 (Hog1), from the rice leaf pathogen Bipolaris oryzae. The deduced amino sequence of the SRM1 gene showed significant homology with Hog1-type MAPK homologues from other phytopathogenic fungi and contained a TGY motif for phosphorylation. The B. oryzae mutants with disruption of the SRM1 gene (Deltasrm1) showed growth inhibition under hyperosmotic, hydrogen peroxide, and UV exposure conditions. The Deltasrm1 mutants showed moderate resistance to dicarboximide and phenylpyrrole fungicides. The Deltasrm1 mutations caused a defect in the expression of the gene that encodes antioxidant enzyme catalase (CAT2) under UV and hyperosmotic conditions. Furthermore, the transcriptional patterns of the three melanin biosynthesis genes (PKS1, THR1, and SCD1) and of a gene of unknown function, uvi-1, which are specifically induced by near-ultraviolet (NUV) radiation, gradually decreased in comparison with the wild-type expression patterns. These results suggest that Srm1 contributes to responses to not only osmostress but also to hydrogen peroxide and UV stress, whereas Srm1 does not appear to regulate directly the expression of genes related to NUV-induced photomorphogenesis. PMID:16553861

  6. Inhibition of p38 mitogen-activated protein kinase potentiates the apoptotic effect of berberine/tumor necrosis factor-related apoptosis-inducing ligand combination therapy

    PubMed Central

    REFAAT, ALAA; ABDELHAMED, SHERIF; SAIKI, IKUO; SAKURAI, HIROAKI

    2015-01-01

    It was previously reported that berberine (BBR) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibited a synergistic apoptotic effect on triple negative breast cancer (TNBC) cells. In addition, the BBR/TRAIL combination treatment sensitized TRAIL-resistant TNBC cells to TRAIL. The aim of the present study was to investigate a novel pathway for enhancing the apoptotic effect of BBR/TRAIL through mitogen-activated protein kinases (MAPKs). Selective inhibitors and small interfering RNAs were utilized to understand the role of p38 MAPK in this pathway. The results demonstrated that p38 MAPK was activated in response to the combination therapy in TRAIL-resistant TNBC cells. In addition, it was revealed that the inhibition of p38 enhanced apoptosis in epidermal growth factor receptor (EGFR)-overexpressing MDA-MB-468 TNBC cells and EGFR-mutant PC-9 non-small-cell lung carcinoma cells, which was associated with the downregulation of EGFR serine phosphorylation. Viability assays for these two cell lines also confirmed the significant reduction of cell viability following p38 inhibition in BBR/TRAIL-treated cells. In conclusion, the present study provided novel evidence for the role of p38 in suppressing BBR/TRAIL-mediated apoptosis and its association with EGFR, which may explain the mechanism of treatment resistance in certain types of cancer. PMID:26622773

  7. Fluorescence polarization-based assays for detecting compounds binding to inactive c-Jun N-terminal kinase 3 and p38α mitogen-activated protein kinase.

    PubMed

    Ansideri, Francesco; Lange, Andreas; El-Gokha, Ahmed; Boeckler, Frank M; Koch, Pierre

    2016-06-15

    Two fluorescein-labeled pyridinylimidazoles were synthesized and evaluated as probes for the binding affinity determination of potential kinase inhibitors to the c-Jun N-terminal kinase 3 (JNK3) and p38α mitogen-activated protein kinase (MAPK). Fluorescence polarization (FP)-based competition binding assays were developed for both enzymes using 1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)-3-(4-((4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)thiourea (5) as an FP probe (JNK3: Kd = 3.0 nM; p38α MAPK: Kd = 5.7 nM). The validation of the assays with known inhibitors of JNK3 and p38α MAPK revealed that both FP assays correlate very well with inhibition data received by the activity assays. This, in addition to the viability of both FP-based binding assays for the high-throughput screening procedure, makes the assays suitable as inexpensive prescreening protocols for JNK3 and p38α MAPK inhibitors. PMID:26954235

  8. Cloning, expression and characterization of a gene encoding mitogen activated protein kinase 2 (MPK2) from Tetrahymena thermophila.

    PubMed

    Arslanyolu, Muhittin; Yıldız, Mehmet Taha

    2014-08-01

    Environmental effects and mitogens determine cell phenotype in eukaryotes mainly through MAPK pathways. However, MAPK signaling pathways in T. thermophila have not been studied comprehensively. This study aims to express recombinant MPK2, a MAPK from T. thermophila, in E. coli to characterize its kinase activity. MPK2 was cloned by RT-PCR using degenerate oligonucleotide primers and RACE method. The full-length cDNA of the MPK2 gene is 1705bp that includes 1281bp ORF coding for a putative protein of 426 amino acids having a mass of 50.2kDa. The putative MPK2 protein contains all eleven conserved subdomains that are characteristics of serine/threonine protein kinases, and a TDY motif, which is a putative dual phosphorylation site common in Protista. MPK2 displays highest 48% overall identity to human ERK5 (MAPK7). The expression vector pGEX4T-1-MPK2 was constructed by inserting the coding region of MPK2 cDNA into pGEX4T-1 after introducing the nine point mutations, and then transformed into E. coli BL21(DE3). Autophosphorylation of 76kDa GST-MPK2 at tyrosine residues was confirmed not only by Western blot using anti-phosphotyrosine monoclonal antibody but also by in vitro kinase assay. GST-MPK2 was also able to phosphorylate the artificial substrate myelin basic protein. This study concludes that the free-living unicellular protist T. thermophila MPK2 has commonly conserved MAPK enzyme features, possibly involved in the regulation of cell survival responding to abiotic or biotic stressors, and the production and movement of haploid gametic nuclei between pairs during conjugation. PMID:24858074

  9. In Silico Elucidation and Inhibition Studies of Selected Phytoligands Against Mitogen-Activated Protein Kinases of Protozoan Parasites.

    PubMed

    Gupta, Chhedi Lal; Akhtar, Salman; Kumar, Nilesh; Ali, Jasarat; Pathak, Neelam; Bajpai, Preeti

    2016-03-01

    Parasitic MAPKs exhibiting significant divergence with humans and playing an imperative role in parasitic metabolic activities have been exploited from several years as important targets for development of novel therapeutics. In addition, the emergence of the drug-resistant variants of parasitic diseases in the recent years has aroused a great need for the development of potent inhibitors against them. In the present study, we selected the metabolically active MAPKs LmxMPK4, PfMAP2 and TbMAPK5 of the three parasitic protozoans Leishmania mexicana, Plasmodium falciparum and Trypanosoma brucei, respectively. The homology modeling technique was used to develop the 3D structures of these proteins, and the same was validated by PROCHECK, ERRAT, ProQ and ProSA web servers to check the reliability. Ten phytoligands were employed for molecular docking studies with these proteins to search for potent phytoligand as a broad spectrum inhibitor. In this regard, two phytoligands (aspidocarpine for LmxMPK4 and TbMAPK5 and cubebin for PfMAP2) were found to be more effective inhibitors, in terms of robust binding energy, strong inhibition constant and better interactions between protein-ligand complexes. Furthermore, predicted ADME and toxicity properties suggested that these identified phytoligands exhibited comparable results to control drugs potentiating them as persuasive therapeutic agents for Leishmania, Trypanosoma and Plasmodium sp. PMID:26264054

  10. Protein–peptide complex crystallization: a case study on the ERK2 mitogen-activated protein kinase

    SciTech Connect

    Gógl, Gergő; Törő, Imre; Reményi, Attila

    2013-03-01

    A rational surface-engineering approach led to the crystal structure determination of ERK2–docking peptide complexes. Linear motifs normally bind with only medium binding affinity (K{sub d} of ∼0.1–10 µM) to shallow protein-interaction surfaces on their binding partners. The crystallization of proteins in complex with linear motif-containing peptides is often challenging because the energy gained upon crystal packing between symmetry mates in the crystal may be on a par with the binding energy of the protein–peptide complex. Furthermore, for extracellular signal-regulated kinase 2 (ERK2) the protein–peptide docking surface is comprised of a small hydrophobic surface patch that is often engaged in the crystal packing of apo ERK2 crystals. Here, a rational surface-engineering approach is presented that involves mutating protein surface residues that are distant from the peptide-binding ERK2 docking groove to alanines. These ERK2 surface mutations decrease the chance of ‘unwanted’ crystal packing of ERK2 and the approach led to the structure determination of ERK2 in complex with new docking peptides. These findings highlight the importance of negative selection in crystal engineering for weakly binding protein–peptide complexes.

  11. Activation of p38 mitogen-activated protein kinase by celecoxib oppositely regulates survivin and gamma-H2AX in human colorectal cancer cells

    SciTech Connect

    Hsiao, P.-W.; Chang, C.-C.; Liu, H.-F.; Tsai, C.-M.; Chiu, Ted H.; Chao, J.-I . E-mail: chaoji@mail.tcu.edu.tw

    2007-07-01

    Cancer cells express survivin that facilitates tumorigenesis. Celecoxib has been shown to reduce human colorectal cancers. However, the role and regulation of survivin by celecoxib in colorectal carcinoma cells remain unclear. Treatment with 40-80 {mu}M celecoxib for 24 h induced cytotoxicity and proliferation inhibition via a concentration-dependent manner in RKO colorectal carcinoma cells. Celecoxib blocked the survivin protein expression and increased the phosphorylation of H2AX at serine-193 ({gamma}-H2AX). The survivin gene knockdown by transfection with a survivin siRNA revealed that the loss of survivin correlated with the expression of {gamma}-H2AX. Meanwhile, celecoxib increased caspase-3 activation and apoptosis. Celecoxib activated the phosphorylation of p38 mitogen-activated protein (MAP) kinase. The phosphorylated proteins of p38 MAP kinase and {gamma}-H2AX were observed in the apoptotic cells. SB203580, a specific p38 MAP kinase inhibitor, protected the survivin protein expression and decreased the levels of {gamma}-H2AX and apoptosis in the celecoxib-exposed cells. The blockade of survivin expression increased the celecoxib-induced cytotoxicity; conversely, overexpression of survivin by transfection with a survivin-expressing vector raised the cancer cell proliferation and resisted the celecoxib-induced cell death. Our results provide for the first time that p38 MAP kinase participates in the down-regulation of survivin and subsequently induces the activation of {gamma}-H2AX for mediating apoptosis following treatment with celecoxib in human colorectal cancer cells.

  12. Interaction of Omega, Sigma, and Theta Glutathione Transferases with p38b Mitogen-Activated Protein Kinase from the Fruit Fly, Drosophila melanogaster

    PubMed Central

    Wongtrakul, J.; Janphen, K.; Saisawang, C.; Ketterman, A.J.

    2014-01-01

    Glutathione S-transferases (GSTs) are a diverse family of phase II detoxification enzymes found in almost all organisms. Besides playing a major role in the detoxification of xenobiotic and toxic compounds, GSTs are also involved in the regulation of mitogen activated protein (MAP) kinase signal transduction by interaction with proteins in the pathway. An in vitro study was performed for Theta, Omega, Sigma GSTs and their interaction with MAP kinase p38b protein from the fruit fly Drosophila melanogaster Meigen (Diptera: Drosophilidae). The study included the effects of all five Omega class GSTs (DmGSTO1, DmGSTO2a, DmGSTO2b, DmGSTO3, DmGSTO4), all five Theta class GSTs (DmGSTT1, DmGSTT2, DmGSTT3a, DmGSTT3b, DmGSTT4), and one Sigma class glutathione transferase on the activity of Drosophila p38b, including the reciprocal effect of this kinase protein on glutathione transferase activity. It was found that DmGSTT2, DmGSTT3b, DmGSTO1, and DmGSTO3 activated p38b significantly. Substrate specificities of GSTs were also altered after co-incubation with p38b. Although p38b activated DmGSTO1, DmGSTO2a, and DmGSTT2, it inhibited DmGSTT3b and DmGSTO3 activity toward xenobiotic and physiological substrates tested. These results suggest a novel link between Omega and Theta GSTs with the p38b MAP kinase pathway. PMID:25373207

  13. Livin enhances tumorigenesis by regulating the mitogen-activated protein kinase signaling pathway in human hypopharyngeal squamous cell carcinoma.

    PubMed

    Kim, Sun-Ae; Yoon, Tae Mi; Lee, Dong Hoon; Lee, Joon Kyoo; Park, Young-Lan; Chung, Ik-Joo; Joo, Young-Eun; Lim, Sang Chul

    2016-07-01

    Livin, a member of the human inhibitor of apoptosis protein (IAP) family, is expressed at high levels in various human cancer tissues and may have prognostic significance. The aim of the present study was to evaluate the effect of Livin on tumor cell behavior and oncogenic signaling pathways in human hypopharyngeal squamous cell carcinoma (HSCC). Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to determine the mRNA and protein expression levels, respectively. A cell proliferation assay and cell cycle analysis were used to assess the functional effects of small interfering RNA‑mediated Livin knockdown. Livin was overexpressed in fresh HSCC tissues, compared with the adjacent normal mucosa. Livin knockdown led to significantly reduced cell proliferation and cell cycle arrest in the G1 phase of the human HSCC cells. The expression levels of c‑myc, cyclin D1, cyclin D3, cyclin‑dependent kinase (CDK)4 and CDK6 were decreased. The phosphorylation levels of extracellular signal‑regulated kinase 1/2, p38, c‑Jun N-terminal kinase and Akt were also decreased by Livin knockdown in the HSCC cells. Taken together, the results of the present study suggested that Livin may enhance tumorigenesis by modulating the mitogen‑activated/Akt signaling pathways in human HSCC. PMID:27175933

  14. Interaction of Snail and p38 mitogen-activated protein kinase results in shorter overall survival of ovarian cancer patients.

    PubMed

    Hipp, Susanne; Berg, Daniela; Ergin, Bilge; Schuster, Tibor; Hapfelmeier, Alexander; Walch, Axel; Avril, Stefanie; Schmalfeldt, Barbara; Höfler, Heinz; Becker, Karl-Friedrich

    2010-12-01

    Epithelial ovarian cancer is a highly metastatic disease and the leading cause of death among cancer of the female genital tract. Abnormal epidermal growth factor receptor (EGFR) signalling has been shown to be involved in epithelial-mesenchymal transition (EMT), an early step during metastasis. Additionally, over-expression of the E-cadherin repressor Snail, a key regulator of EMT, has previously been found to be associated with unfavourable prognostic features. Thus, the aim of our study was to elucidate the role of EGFR-dependent signalling pathways for Snail expression in ovarian cancer. For this purpose, we analysed 25 formalin-fixed and paraffin-embedded (FFPE) primary tumours and their corresponding metastases for the expression of 25 signalling pathway molecules by reverse phase protein arrays. We found a significant correlation of Snail with EGFR((Tyr1086)) and p38 MAPK((Thr180/Tyr182)) in primary ovarian carcinoma and with EGFR((Tyr1086)) in their corresponding metastasis. Additionally, we showed that high expression levels of Snail in primary tumours combined with high expression levels of the phosphorylated p38 MAPK((Thr180/Tyr182)) in metastasis lead to an increased risk for death in ovarian carcinoma patients. Thus, for future combinatorial cancer therapy, drug combinations that best target the deregulated protein network in each individual patient should be selected. PMID:20957493

  15. Astragalus polysaccharide upregulates hepcidin and reduces iron overload in mice via activation of p38 mitogen-activated protein kinase.

    PubMed

    Ren, Feng; Qian, Xin-Hua; Qian, Xin-Lai

    2016-03-25

    Thalassemia is a genetic disease characterized by iron overload which is a major detrimental factor contributing to mortality and organ damage. The hepcidin secreted by liver plays an essential role in orchestrating iron metabolism. Lowering iron load in thalassemia patients by means of increasing hepcidin might be a therapeutic strategy. In this study, we first found that astragalus polysaccharide (APS) significantly increased hepcidin expression in HepG2 and L-02 cell lines originating from hepatocytes and mice liver, respectively. Following treatment with APS, the iron concentrations in serum, liver, spleen, and heart were significantly reduced in comparison to saline treated control mice. In further experiments, upregulation of interleukin-6 (IL-6) and enhanced p38 MAPK phosphorylation were detected in APS treated cells and mice, and as documented in previous studies, IL-6 and P38 MAPK phosphorylation are involved in the regulation of hepcidin expression. We also found that the effects of APS on upregulating hepcidin and IL-6 expressions could be antagonized by pretreatment with SB203580, an inhibitor of p38 MAPK signaling. These findings suggest that activation of p38 MAPK and release of IL-6 might mediate induction of hepcidin by APS. It is concluded that APS might have therapeutic implications in patients with iron overload, especially for thalassemia patients. PMID:26915800

  16. The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides.

    PubMed

    Zhang, Yueping; Choi, Yoon-E; Zou, Xuexiao; Xu, Jin-Rong

    2011-02-01

    Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity. PMID:20887797

  17. Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase.

    PubMed Central

    Conway, A M; Rakhit, S; Pyne, S; Pyne, N J

    1999-01-01

    The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated- protein-kinase (p42/p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric G-protein Gi) induced an approx. 40-50% decrease in the activation of c-Src and p42/p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PP1, which abolished p42/p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and p42/p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, Gi, to regulate c-Src and subsequent p42/p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in p42/p44 MAPK activation. This is confirmed here by experiments which showed that PI3K inhibitors (wortmannin and LY294002) reduced the activation of p42/p44 MAPK by PDGF. PI3K activity was increased in Grb-2 immunoprecipitates from PDGF-stimulated cells and was decreased by pretreating these cells with PTX. These findings show that Gi might also promote Grb-2-PI3K complex formation and that Grb-2 may be a site at which PI3K is integrated into the p42/p44 MAPK cascade. In conclusion, our results demonstrate that Gi enables the PDGFR to signal more efficiently to p42/p44 MAPK, and this appears to be achieved through the regulation of c-Src and Grb-2/PI3K, which are intermediates in the p42/p44 MAPK cascade. PMID:9882612

  18. Novel Antiplatelet Activity of Minocycline Involves Inhibition of MLK3-p38 Mitogen Activated Protein Kinase Axis

    PubMed Central

    Jackson, Joseph W.; Singh, Meera V.; Singh, Vir B.; Jones, Letitia D.; Davidson, Gregory A.; Ture, Sara; Morrell, Craig N.; Schifitto, Giovanni; Maggirwar, Sanjay B.

    2016-01-01

    Platelets play an essential role in hemostasis and wound healing by facilitating thrombus formation at sites of injury. Platelets also mediate inflammation and contain several pro-inflammatory molecules including cytokines and chemokines that mediate leukocyte recruitment and activation. Not surprisingly, platelet dysfunction is known to contribute to several inflammatory disorders. Antiplatelet therapies, such as aspirin, adenosine diphosphate (ADP) antagonists, glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors, and anticoagulants such as warfarin, dampen platelet activity at the risk of unwarranted bleeding. Thus, the development of drugs that reduce platelet-mediated inflammation without interfering with thrombus formation is of importance to combat platelet-associated disorders. We have shown here for the first time that the tetracycline antibiotic, minocycline, administered to HIV-infected individuals reduces plasma levels of soluble CD40L and platelet factor 4 levels, host molecules predominately released by platelets. Minocycline reduced the activation of isolated platelets in the presence of the potent platelet activator, thrombin, as measured by ELISA and flow cytometry. Platelet degranulation was reduced upon exposure to minocycline as shown by mepacrine retention and flow cytometry. However, minocycline had no effect on spreading, aggregation, GPIIb/IIIa activation, or in vivo thrombus formation. Lastly, immunoblot analysis suggests that the antiplatelet activity of minocycline is likely mediated by inhibition of mixed lineage kinase 3 (MLK3)-p38 MAPK signaling axis and loss of p38 activity. Our findings provide a better understanding of platelet biology and a novel repurposing of an established antibiotic, minocycline, to specifically reduce platelet granule release without affecting thrombosis, which may yield insights in generating novel, specific antiplatelet therapies. PMID:27270236

  19. Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

    PubMed

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

    p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

  20. The Response Regulator RRG-1 Functions Upstream of a Mitogen-activated Protein Kinase Pathway Impacting Asexual Development, Female Fertility, Osmotic Stress, and Fungicide Resistance in Neurospora crassa

    PubMed Central

    Jones, Carol A.; Greer-Phillips, Suzanne E.

    2007-01-01

    Two-component systems, consisting of proteins with histidine kinase and/or response regulator domains, regulate environmental responses in bacteria, Archaea, fungi, slime molds, and plants. Here, we characterize RRG-1, a response regulator protein from the filamentous fungus Neurospora crassa. The cell lysis phenotype of Δrrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protein kinase (MAPK) pathway: os-4 (MAPK kinase kinase), os-5 (MAPK kinase), and os-2 (MAPK). Similar to os mutants, Δrrg-1 strains are sensitive to hyperosmotic conditions, and they are resistant to the fungicides fludioxonil and iprodione. Like os-5, os-4, and os-2 mutants, but in contrast to nik-1/os-1 strains, Δrrg-1 mutants do not produce female reproductive structures (protoperithecia) when nitrogen starved. OS-2-phosphate levels are elevated in wild-type cells exposed to NaCl or fludioxonil, but they are nearly undetectable in Δrrg-1 strains. OS-2-phosphate levels are also low in Δrrg-1, os-2, and os-4 mutants under nitrogen starvation. Analysis of the rrg-1D921N allele, mutated in the predicted phosphorylation site, provides support for phosphorylation-dependent and -independent functions for RRG-1. The data indicate that RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and protoperithecial development through regulation of the OS-4/OS-5/OS-2 MAPK pathway. PMID:17392518

  1. Diphenylarsinic Acid Induced Activation of Cultured Rat Cerebellar Astrocytes: Phosphorylation of Mitogen-Activated Protein Kinases, Upregulation of Transcription Factors, and Release of Brain-Active Cytokines.

    PubMed

    Negishi, Takayuki; Matsumoto, Mami; Kojima, Mikiya; Asai, Ryota; Kanehira, Tomoko; Sakaguchi, Fumika; Takahata, Kazuaki; Arakaki, Rina; Aoyama, Yohei; Yoshida, Hikari; Yoshida, Kenji; Yukawa, Kazunori; Tashiro, Tomoko; Hirano, Seishiro

    2016-03-01

    Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow. PMID:26645585

  2. Two mitogen-activated protein kinase kinases, MKK1 and MEK2, are involved in wounding- and specialist lepidopteran herbivore Manduca sexta-induced responses in Nicotiana attenuata

    PubMed Central

    Heinrich, Maria; Baldwin, Ian T.; Wu, Jianqiang

    2011-01-01

    In a wild tobacco plant, Nicotiana attenuata, two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), play central roles in modulating herbivory-induced phytohormone and anti-herbivore secondary metabolites. However, the identities of their upstream MAPK kinases (MAPKKs) were elusive. Ectopic overexpression studies in N. benthamiana and N. tabacum suggested that two MAPKKs, MKK1 and MEK2, may activate SIPK and WIPK. The homologues of MKK1 and MEK2 were cloned in N. attenuata (NaMKK1 and NaMEK2) and a virus-induced gene silencing approach was used to knock-down the transcript levels of these MAPKK genes. Plants silenced in NaMKK1 and NaMEK2 were treated with wounding or simulated herbivory by applying the oral secretions of the specialist herbivore Manduca sexta to wounds. MAPK activity assay indicated that after wounding or simulated herbivory NaMKK1 is not required for the phosphorylation of NaSIPK and NaWIPK; in contrast, NaMEK2 and other unknown MAPKKs are important for simulated herbivory-elicited activation of NaSIPK and NaWIPK, and after wounding NaMEK2 probably does not activate NaWIPK but plays a minor role in activating NaSIPK. Consistently, NaMEK2 and certain other MAPKKs, but not NaMKK1, are needed for wounding- and simulated herbivory-elicited accumulation of jasmonic acid (JA), JA–isoleucine, and ethylene. Furthermore, both NaMEK2 and NaMKK1 regulate the levels of trypsin proteinase inhibitors. The findings underscore the complexity of MAPK signalling pathways and highlight the importance of MAPKKs in regulating wounding- and herbivory-induced responses. PMID:21610019

  3. Herpes Simplex Virus Type 1 ICP27 Induces p38 Mitogen-Activated Protein Kinase Signaling and Apoptosis in HeLa Cells▿

    PubMed Central

    Gillis, Peter A.; Okagaki, Laura H.; Rice, Stephen A.

    2009-01-01

    The herpes simplex virus type 1 (HSV-1) protein ICP27 has been implicated in a variety of functions important for viral replication including host shutoff, viral gene expression, activation of mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK), and apoptosis inhibition. In the present study we sought to examine the functions of ICP27 in the absence of viral infection by creating stable HeLa cell lines that inducibly express ICP27. Here, we characterize two such cell lines and show that ICP27 expression is associated with a cellular growth defect. The observed defect is caused at least in part by the induction of apoptosis as indicated by caspase-3 activation, annexin V staining, and characteristic changes in cellular morphology. In an effort to identify the function of ICP27 responsible for inducing apoptosis, we show that ICP27 expression is sufficient to activate p38 signaling to a level that is similar to that observed during wild-type HSV-1 infection. However, ICP27 expression alone is unable to lead to a strong activation of JNK signaling. Using chemical inhibitors, we show that the ICP27-mediated activation of p38 signaling is responsible for the observed induction of apoptosis in the induced cell lines. Our findings suggest that during viral infection, ICP27 activates p38 and JNK signaling pathways via two distinct mechanisms. ICP27 directly activates p38 signaling, leading to stimulation of the host cell apoptotic pathways. In contrast, robust activation of JNK signaling by ICP27 requires one or more delayed early or late viral gene products and may be associated with the inhibition of apoptosis. PMID:19073744

  4. Downregulation of the Ras–Mitogen-Activated Protein Kinase Pathway by the EphB2 Receptor Tyrosine Kinase Is Required for Ephrin-Induced Neurite Retraction

    PubMed Central

    Elowe, Sabine; Holland, Sacha J.; Kulkarni, Sarang; Pawson, Tony

    2001-01-01

    Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones. PMID:11585923

  5. Luteolin is a bioflavonoid that attenuates adipocyte-derived inflammatory responses via suppression of nuclear factor-κB/mitogen-activated protein kinases pathway

    PubMed Central

    Nepali, Sarmila; Son, Ji-Seon; Poudel, Barun; Lee, Ji-Hyun; Lee, Young-Mi; Kim, Dae-Ki

    2015-01-01

    Background: Inflammation of adipocytes has been a therapeutic target for treatment of obesity and metabolic disorders which cause insulin resistance and hence lead to type II diabetes. Luteolin is a bioflavonoid with many beneficial properties such as antioxidant, antiproliferative, and anti-cancer. Objectives: To elucidate the potential anti-inflammatory response and the underlying mechanism of luteolin in 3T3-L1 adipocytes. Materials and Methods: We stimulated 3T3-L1 adipocytes with the mixture of tumor necrosis factor-α, lipopolysaccharide, and interferon-γ (TLI) in the presence or absence of luteolin. We performed Griess’ method for nitric oxide (NO) production and measure mRNA and protein expressions by real-time polymerase chain reaction and western blotting, respectively. Results: Luteolin opposed the stimulation of inducible nitric oxide synthase and NO production by simultaneous treatment of adipocytes with TLI. Furthermore, it reduced the pro-inflammatory genes such as cyclooxygenase-2, interleukin-6, resistin, and monocyte chemoattractant protein-1. Furthermore, luteolin improved the insulin sensitivity by enhancing the expression of insulin receptor substrates (IRS1/2) and glucose transporter-4 via phosphatidylinositol-3K signaling pathway. This inhibition was associated with suppression of Iκ-B-α degradation and subsequent inhibition of nuclear factor-κB (NF-κB) p65 translocation to the nucleus. In addition, luteolin blocked the phosphorylation of ERK1/2, c-Jun N-terminal Kinases and also p38 mitogen-activated protein kinases (MAPKs). Conclusions: These results illustrate that luteolin attenuates inflammatory responses in the adipocytes through suppression of NF-κB and MAPKs activation, and also improves insulin sensitivity in 3T3-L1 cells, suggesting that luteolin may represent a therapeutic agent to prevent obesity-associated inflammation and insulin resistance. PMID:26246742

  6. Delta Opioid activation of the Mitogen-activated protein kinase cascade does not require transphosphorylation of Receptor Tyrosine Kinases

    PubMed Central

    Kramer, H Kenneth; Onoprishvili, Irma; Andria, Matthew L; Hanna, Kayane; Sheinkman, Karina; Haddad, Lisa B; Simon, Eric J

    2002-01-01

    Background In this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned δ-opioid receptor (δ-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive. Results Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed δ-ORs are stimulated by the δ-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the δ-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In δ-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation. Conclusion Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated δ-OR, itself, is likely to act as its own signalling scaffold. PMID:11897012

  7. The mitogen-activated protein kinome from Anopheles gambiae: identification, phylogeny and functional characterization of the ERK, JNK and p38 MAP kinases

    PubMed Central

    2011-01-01

    Background Anopheles gambiae is the primary mosquito vector of human malaria parasites in sub-Saharan Africa. To date, three innate immune signaling pathways, including the nuclear factor (NF)-kappaB-dependent Toll and immune deficient (IMD) pathways and the Janus kinase/signal transducers and activators of transcription (Jak-STAT) pathway, have been extensively characterized in An. gambiae. However, in addition to NF-kappaB-dependent signaling, three mitogen-activated protein kinase (MAPK) pathways regulated by JNK, ERK and p38 MAPK are critical mediators of innate immunity in other invertebrates and in mammals. Our understanding of the roles of the MAPK signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted. Results In this study, we present the orthologs and phylogeny of 17 An. gambiae MAPKs, two of which were previously unknown and two others that were incompletely annotated. We also provide detailed temporal activation profiles for ERK, JNK, and p38 MAPK in An. gambiae cells in vitro to immune signals that are relevant to malaria parasite infection (human insulin, human transforming growth factor-beta1, hydrogen peroxide) and to bacterial lipopolysaccharide. These activation profiles and possible upstream regulatory pathways are interpreted in light of known MAPK signaling cascades. Conclusions The establishment of a MAPK "road map" based on the most advanced mosquito genome annotation can accelerate our understanding of host-pathogen interactions and broader physiology of An. gambiae and other mosquito species. Further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the MAP kinase cascades and other networked pathways will facilitate identification of the

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

    PubMed

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

    2013-04-24

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

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

    PubMed Central

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

    2013-01-01

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

  10. Bombesin stimulates cholecystokinin secretion through mitogen-activated protein-kinase-dependent and -independent mechanisms in the enteroendocrine STC-1 cell line.

    PubMed Central

    Némoz-Gaillard, E; Cordier-Bussat, M; Filloux, C; Cuber, J C; Van Obberghen, E; Chayvialle, J A; Abello, J

    1998-01-01

    Bombesin has been reported to stimulate cholecystokinin (CCK) secretion from rat duodeno-jejunal I-cells. Bombesin was shown to activate mitogen-activated protein kinases (MAPKs) in cell types such as Swiss 3T3 fibroblasts and rat pancreatic acinar cells. No information is available on whether MAPK is activated in intestinal endocrine cells upon bombesin stimulation. This was studied by using the CCK-producing enteroendocrine cell line STC-1. Bombesin stimulated markedly and transiently both p42(MAPK) and p44(MAPK), with a maximum at 2 min, and a decrease to basal levels within 10 min. As expected, bombesin stimulated MAPK kinase 1 (MEK-1) activity. Activation of protein kinase C (PKC) with PMA also stimulated p42(MAPK), p44(MAPK) and MEK-1. Treatment of cells with PD 098059 (at 10 microM or 30 microM), which selectively inhibits MEK phosphorylation, blocked bombesin-induced p42(MAPK) and p44(MAPK) activation for at least 90 min. However, PD 098059 inhibited bombesin- and PMA-stimulated CCK secretion during the first 15 min, but failed to significantly reduce CCK release at later times. Inhibition of PKC with staurosporine, or PKC down-regulation by prolonged treatment with PMA, both drastically decreased MEK-1, p42(MAPK) and p44(MAPK) activation upon bombesin stimulation. Additionally, PKC activation appeared to be required for both MAPK-dependent (early) and -independent (late) CCK responses to bombesin. It is concluded that the early CCK secretory response of STC-1 cells to bombesin involves MAPK pathway activation through a PKC-dependent mechanism, whereas the late phase of bombesin-induced CCK secretion, that also requires PKC, appears to result from a MAPK-independent process. PMID:9512470

  11. A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone.

    PubMed Central

    Roberson, M S; Misra-Press, A; Laurance, M E; Stork, P J; Maurer, R A

    1995-01-01

    Gonadotropin-releasing hormone (GnRH) interacts with a G protein-coupled receptor and increases the transcription of the glycoprotein hormone alpha-subunit gene. We have explored the possibility that mitogen-activated protein kinase (MAPK) plays a role in mediating GnRH effects on transcription. Activation of the MAPK cascade by an expression vector for a constitutively active form of the Raf-1 kinase led to stimulation of the alpha-subunit promoter in a concentration-dependent manner. GnRH treatment was found to increase the phosphorylation of tyrosine residues of MAPK and to increase MAPK activity, as determined by an immune complex kinase assay. A reporter gene assay using the MAPK-responsive, carboxy-terminal domain of the Elk1 transcription factor was also consistent with GnRH-induced activation of MAPK. Interference with the MAPK pathway by expression vectors for kinase-defective MAPKs or vectors encoding MAPK phosphatases reduced the transcription-stimulating effects of GnRH. The DNA sequences which are required for responses to GnRH include an Ets factor-binding site. An expression vector for a dominant negative form of Ets-2 was able to reduce GnRH effects on expression of the alpha-subunit gene. These findings provide evidence that GnRH treatment leads to activation of the MAPK cascade in gonadotropes and that activation of MAPK contributes to stimulation of the alpha-subunit promoter. It is likely that an Ets factor serves as a downstream transcriptional effector of MAPK in this system. PMID:7791760

  12. The mitogen-activated protein kinase pathway plays a critical role in regulating immunological properties of BRAF mutant cutaneous melanoma cells.

    PubMed

    Whipple, Chery A; Boni, Andrea; Fisher, Jan L; Hampton, Thomas H; Tsongalis, Gregory J; Mellinger, Diane L; Yan, Shaofeng; Tafe, Laura J; Brinckerhoff, Constance E; Turk, Mary J; Mullins, David W; Fadul, Camilo E; Ernstoff, Marc S

    2016-06-01

    The advent of drugs targeting the mitogen-activated protein kinase (MAPK) pathway has markedly changed the treatment of advanced-stage melanoma harboring BRAF mutations. However, drug resistance, through mechanisms not well elucidated, often occurs. A better understanding of how melanoma-derived immunologically active molecules change in response to MAPK inhibition of BRAF mutated (BRAF) and BRAF wild type (BRAF) melanomas could help identify promising treatment combinations of small molecule inhibitors and immunotherapy. To this aim, we treated 13 BRAF and 13 BRAF mutated human melanoma cell lines with either a specific BRAF inhibitor or an MEK1/2 inhibitor and analyzed changes in the secretion of 42 selected cytokines, chemokines, and growth factors. We also measured changes in the expression levels of immunologically relevant melanoma cell surface markers. The BRAF melanomas showed minimal changes in response to the inhibitors, whereas the BRAF cell lines showed, on average, a significant decrease in IFNα2, interleukin-7, Fractalkine, GCSF, GRO, TGFα2, interleukin-8, and VEGF, as well as a reduction in pERK and pMEK protein levels, upon MAPK pathway blockade. BRAF inhibition in BRAF cell lines also resulted in significant changes in the expression of several surface markers including upregulation of β2-microglobulin as well as a decrease in MIC A/B and TRAIL-R2. These results indicate that MAPK pathway inhibition leads to changes in the immunological properties of mutant BRAF melanoma cells and lends support for future studies aimed at designing effective treatment strategies that combine BRAF and MEK inhibition with immunotherapy. PMID:26974965

  13. Albumin stimulates p44/p42 extracellular-signal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells.

    PubMed

    Dixon, R; Brunskill, N J

    2000-03-01

    The presence of protein in the urine of patients with renal disease is an adverse prognostic feature. It has therefore been suggested that proteinuria per se may be responsible for the development of renal tubulo-interstitial scarring and fibrosis, and disturbances in tubular cell growth and proliferation. We have used the opossum kidney proximal tubular cell line to investigate the effects of albumin on cell growth. The effect of albumin on cell proliferation was investigated by cell counting and measurement of [(3)H]thymidine incorporation. We studied the effect of recombinant human albumin on the activity of p44/p42 extracellular-signal-regulated mitogen-activated protein kinase (MAP kinase ) using an in vitro kinase assay, and immunoblotting with antibodies against active extracellular-signal-regulated kinase (ERK). The effects of the ERK inhibitor PD98059 were also examined. Recombinant human albumin was found to stimulate proliferation of opossum kidney cells in a dose-dependent manner, with maximal stimulation at a concentration of 1 mg/ml. In addition, recombinant human albumin activated ERK in a time-dependent (maximal after 5 min) and dose-dependent (maximal at 1 mg/ml) fashion. These effects on cell proliferation and ERK activity were inhibited by PD98059, and were not reproduced by ovalbumin or mannitol. The data therefore indicate that albumin is able to stimulate growth and proliferation of proximal tubular cells that is dependent on the ERK family of MAP kinases. The potential importance of this pathway in the development of renal disease is discussed. PMID:10677388

  14. Disease Resistance and Abiotic Stress Tolerance in Rice Are Inversely Modulated by an Abscisic Acid–Inducible Mitogen-Activated Protein KinaseW⃞

    PubMed Central

    Xiong, Lizhong; Yang, Yinong

    2003-01-01

    Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in eukaryotic organisms. However, little is known about the role of MAPKs in modulating the interaction of defense pathways activated by biotic and abiotic factors. In this study, we have isolated and functionally characterized a stress-responsive MAPK gene (OsMAPK5) from rice. OsMAPK5 is a single-copy gene but can generate at least two differentially spliced transcripts. The OsMAPK5 gene, its protein, and kinase activity were inducible by abscisic acid as well as various biotic (pathogen infection) and abiotic (wounding, drought, salt, and cold) stresses. To determine its biological function, we generated and analyzed transgenic rice plants with overexpression (using the 35S promoter of Cauliflower mosaic virus) or suppression (using double-stranded RNA interference [dsRNAi]) of OsMAPK5. Interestingly, suppression of OsMAPK5 expression and its kinase activity resulted in the constitutive expression of pathogenesis-related (PR) genes such as PR1 and PR10 in the dsRNAi transgenic plants and significantly enhanced resistance to fungal (Magnaporthe grisea) and bacterial (Burkholderia glumae) pathogens. However, these same dsRNAi lines had significant reductions in drought, salt, and cold tolerance. By contrast, overexpression lines exhibited increased OsMAPK5 kinase activity and increased tolerance to drought, salt, and cold stresses. These results strongly suggest that OsMAPK5 can positively regulate drought, salt, and cold tolerance and negatively modulate PR gene expression and broad-spectrum disease resistance. PMID:12615946

  15. The food contaminant deoxynivalenol activates the mitogen activated protein kinases in the intestine: interest of ex vivo models as an alternative to in vivo experiments.

    PubMed

    Lucioli, Joelma; Pinton, Philippe; Callu, Patrick; Laffitte, Joëlle; Grosjean, François; Kolf-Clauw, Martine; Oswald, Isabelle P; Bracarense, Ana Paula F R L

    2013-05-01

    Trichothecenes induce changes in the intestinal barrier function through decreased expression of cell junction proteins and apoptosis of enterocytes. The mitogen activated protein kinases (MAPK) play an important role in the signaling pathways of cell turnover and differentiation. Using ex vivo and in vivo approaches, the purpose of this study was to investigate the ability of low doses of DON to induce histological changes in the intestine and to activate the MAPK ERK 1/2, p38 and JNK. Twelve weaning piglets received during four weeks a control diet or a DON-contaminated diet (2.3 mg DON/kg feed). Six weaning piglets were used to prepare jejunal explants (ex vivo model). Explants were exposed during 4 h to vehicle, 5 or 10 μM DON. Intestinal changes were graded using a histological score. Pigs fed a DON-diet and explants exposed to DON showed a significant decrease in the jejunal score. In both models, the toxin significantly enhanced phosphorylation of ERK 1/2 and p38, whereas the increased phosphorylation of JNK was non significant. Taken together these results indicate that in vivo or ex vivo exposure of intestinal tissue to DON lead to similar intestinal lesions and activation of MAPK. These effects could impair the homeostasis of intestinal tissue in the aspects of barrier function and immune protection. The similarity of the in vivo and ex vivo results provides also strong evidence that the jejunal explant model is a good alternative for toxicological studies in intestinal tissue. PMID:23403092

  16. Octacalcium phosphate crystals directly stimulate expression of inducible nitric oxide synthase through p38 and JNK mitogen-activated protein kinases in articular chondrocytes

    PubMed Central

    Ea, Hang-Korng; Uzan, Benjamin; Rey, Christian; Lioté, Frédéric

    2005-01-01

    Basic calcium phosphate (BCP) crystals, including hydroxyapatite, octacalcium phosphate (OCP) and carbonate-apatite, have been associated with severe osteoarthritis and several degenerative arthropathies. Most studies have considered the chondrocyte to be a bystander in the pathogenesis of calcium crystal deposition disease, assuming that synovial cell cytokines were the only triggers of chondrocyte activation. In the present study we identified direct activation of articular chondrocytes by OCP crystals, which are the BCP crystals with the greatest potential for inducing inflammation. OCP crystals induced nitric oxide (NO) production and inducible nitric oxide synthase (NOS) mRNA expression by isolated articular chondrocytes and cartilage fragments, in a dose-dependent manner and with variations over time. OCP crystals also induced IL-1β mRNA expression. Using pharmacological and cytokine inhibitors, we observed that OCP crystals induced NO production and inducible NOS mRNA activation were regulated at both the transcriptional and the translational levels; were independent from IL-1β gene activation; and involved p38 and c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, as further confirmed by OCP crystal-induced p38 and JNK MAPK phosphorylation. Taken together, our data suggest that the transcriptional inducible NOS response to OCP crystals involved both the p38 and the JNK MAPK pathways, probably under the control of activator protein-1. NO, a major mediator of cartilage degradation, can be directly produced by BCP crystals in chondrocytes. Together with synovial activation, this direct mechanism may be important in the pathogenesis of destructive arthropathies triggered by microcrystals. PMID:16207333

  17. Effect of training on activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in rat soleus muscle.

    PubMed

    Lee, Jong Sam; Bruce, Clinton R; Spurrell, Brian E; Hawley, John A

    2002-08-01

    1. The effect of a chronic programme of either low- or moderate-to-high-intensity treadmill running on the activation of the extracellular-signal regulated protein kinase (ERK1/2) and the p38 mitogen-activated protein kinase (MAPK) pathways was determined in rat muscle. 2. Sprague-Dawley rats were assigned to one of three groups: (i) sedentary (NT; n = 8); (ii) low-intensity training (8 m/min; LIT; n = 16); and (iii) moderate-to-high-intensity training (28 m/min; HIT; n = 16). The training regimens were planned so that animals covered the same distance and had similar glycogen utilization for both LIT and HIT exercise sessions. 3. A single bout of LIT or HIT following 8 weeks of training led to a twofold increase in the phosphorylation of ERK1/2 (P = 0.048) and a two- to threefold increase in p38 MAPK (P = 0.005). Extracellular signal-regulated kinase 1/2 phosphorylation in muscle sampled 48 h after the last exercise bout was similar to sedentary values, while p38 MAPK phosphorylation was 70-80% lower than sedentary. One bout of LIT or HIT increased total ERK1/2 and p38 MAPK expression, with the magnitude of this increase being independent of prior exercise intensity or duration. Extracellular signal- regulated kinase 1/2 expression was increased three- to fourfold in muscle sampled 48 h after the last exercise bout irrespective of the prior training programme (P = 0.027), but p38 MAPK expression was approximately 90% lower than sedentary values. 4. In conclusion, exercise-training of different intensities/ durations results in selective postexercise activation of intracellular signalling pathways, which may be one mechanism regulating specific adaptations induced by diverse training programmes. PMID:12099995

  18. All-trans retinoic acid modulates mitogen-activated protein kinase pathway activation in human scleral fibroblasts through retinoic acid receptor beta

    PubMed Central

    Huo, Lijun; Cui, Dongmei; Yang, Xiao; Gao, Zhenya; Trier, Klaus

    2013-01-01

    Purpose All-trans retinoic acid (ATRA) is known to inhibit the proliferation of human scleral fibroblasts (HSFs) and to modulate the scleral intercellular matrix composition, and may therefore serve as a mediator for controlling eye growth. Cell proliferation is regulated by the mitogen-activated protein kinase (MAPK) pathway. The aim of the current study was to investigate whether changed activation of the MAPK pathway could be involved in the response of HSFs exposed to ATRA. Methods HSFs were cultured in Dulbecco Modified Eagle's Medium/F12 (DMEM/F12) and exposed to 1 μmol/l ATRA for 10 min, 30 min, 1 h, 8 h, or 24 h. The activation of extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun N-terminal kinase (JNK) in HSFs was assessed with western blot analysis and immunocytofluorescence. Results After exposure to ATRA for 24 h, the HSFs appeared shrunken and thinner than the control cells. The intercellular spaces were wider, and the HSFs appeared less numerous than in the control culture. Western blot showed decreased activation of ERK 1/2 in the HSFs from 30 min (p=0.01) to 24 h (p<0.01) after the start of exposure to ATRA, and increased activation of the JNK protein from 10 to 30 min (p<0.01) after the start of exposure to ATRA. Indirect immunofluorescence confirmed changes in activation of ERK 1/2 and JNK in HSFs exposed to ATRA. No change in activation of p38 in HSFs was observed after exposure to ATRA. Pretreatment of the HSFs with LE135, an antagonist of retinoic acid receptor beta (RARβ), abolished the ATRA-induced changes inactivation of ERK 1/2 and JNK. Conclusions ATRA inhibits HSF proliferation by a mechanism associated with modulation of ERK 1/2 and JNK activation and depends on stimulation of retinoic acid receptor beta. PMID:23946634

  19. The functional synergy between IL-12 and IL-2 involves p38 mitogen-activated protein kinase and is associated with the augmentation of STAT serine phosphorylation.

    PubMed

    Gollob, J A; Schnipper, C P; Murphy, E A; Ritz, J; Frank, D A

    1999-04-15

    IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-gamma, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways. PMID:10201984

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

    PubMed Central

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

    1994-01-01

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

  1. Gamma-hydroxybutyrate reduces mitogen-activated protein kinase phosphorylation via GABA B receptor activation in mouse frontal cortex and hippocampus.

    PubMed

    Ren, Xiuhai; Mody, Istvan

    2003-10-24

    gamma-Hydroxybutyrate (GHB) naturally occurs in the brain, but its exogenous administration induces profound effects on the central nervous system in animals and humans. The intracellular signaling mechanisms underlying its actions remain unclear. In the present study, the effects of GHB on the activation (phosphorylation) of mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase 1 and 2 (ERK1/2), were investigated. Acute administration of GHB (500 mg/kg, intraperitoneal) induced a fast and long lasting inhibition of MAP kinase phosphorylation in both frontal cortex and hippocampus. The reduced MAP kinase phosphorylation was observed in the CA1 and CA3 areas but not in the dentate gyrus. Pretreatment with the specific gamma-aminobutyric acid, type B (GABAB), receptor antagonist CGP56999A (20 mg/kg, intraperitoneal) prevented the action of GHB, and the effect of GHB was mimicked by baclofen, a selective GABAB receptor agonist, whereas the high affinity GHB receptor antagonist NCS-382 (200 mg/kg, intraperitoneal) had no effect on GHB-inhibited MAP kinase phosphorylation. Moreover, the GHB dehydrogenase inhibitor valproate (500 mg/kg, intraperitoneal), which inhibits the conversion of GHB into GABA, failed to block the effect of GHB on MAP kinase phosphorylation. Altogether, these data suggest that GHB, administered in vivo, reduces MAP kinase phosphorylation via a direct activation of GABAB receptors by GHB. In contrast, GHB (10 mm for 15 min) was found ineffective on MAP kinase phosphorylation in brain slices, indicating important differences in the conditions required for the second messenger activating action of GHB. PMID:12923192

  2. Two guard cell mitogen-activated protein kinases, MPK9 and MPK12, function in methyl jasmonate-induced stomatal closure in Arabidopsis thaliana.

    PubMed

    Khokon, Md A R; Salam, M A; Jammes, F; Ye, W; Hossain, M A; Uraji, M; Nakamura, Y; Mori, I C; Kwak, J M; Murata, Y

    2015-09-01

    Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9-1 and mpk12-1 single mutants as well as wild-type plants, but not in mpk9-1 mpk12-1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA-induced stomatal closure in wild-type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9-1, mpk12-1 and mpk9-1 mpk12-1 mutants, as well in wild-type plants. Furthermore, MeJA triggered elevation of cytosolic Ca(2+) concentration ([Ca(2+)]cyt ) in the mpk9-1 mpk12-1 double mutant as well as wild-type plants. Activation of S-type anion channels by MeJA was impaired in mpk9-1 mpk12-1. Together, these results indicate that MPK9 and MPK12 function upstream of S-type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca(2+)]cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells. PMID:25703019

  3. p38γ Mitogen-Activated Protein Kinase Contributes to Oncogenic Properties Maintenance and Resistance to Poly (ADP-Ribose)-Polymerase-1 Inhibition in Breast Cancer12

    PubMed Central

    Meng, Fanyan; Zhang, Haijun; Liu, Gang; Kreike, Bas; Chen, Wei; Sethi, Seema; Miller, Fred R; Wu, Guojun

    2011-01-01

    p38γ MAPK, one of the four members of p38 mitogen-activated protein kinases (MAPKs), has previously been shown to harbor oncogenic functions. However, the biologic function of p38γ MAPK in breast cancer has not been well defined. In this study, we have shown that p38γ MAPK is overexpressed in highly metastatic human and mouse breast cancer cell lines and p38γ MAPK expression is preferentially associated with basal-like and metastatic phenotypes of breast tumor samples. Ectopic expression of p38γ MAPK did not lead to an increase in oncogenic properties in vitro in most tested mammary epithelial cells. However, knockdown of p38γ MAPK expression resulted in a dramatic decrease in cell proliferation, colony formation, cell migration, invasion in vitro and significant retardation of tumorigenesis, and long-distance metastasis to the lungs in vivo. Moreover, knockdown of p38γ MAPK triggered the activation of AKT signaling. Inhibition of this feedback loop with various PI3K/AKT signaling inhibitors facilitated the effect of targeting p38γ MAPK. We further found that overexpression of p38γ MAPK did not promote cell resistance to chemotherapeutic agents doxorubicin and paclitaxel but significantly increased cell resistance to PJ-34, a DNA damage agent poly (ADP-ribose)-polymerase-1 (PARP) inhibitor in vitro and in vivo. Finally, we identified that p38γ MAPK overexpression led to marked cell cycle arrest in G2/M phase. Our study for the first time clearly demonstrates that p38γ MAPK is a promising target for the design of targeted therapies for basal-like breast cancer with metastatic characteristics and for overcoming potential resistance against the PARP inhibitor. PMID:21532888

  4. Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

    PubMed Central

    Rausch, O; Marshall, C J

    1997-01-01

    The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK. PMID:9032244

  5. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

    PubMed Central

    Reszka, A A; Bulinski, J C; Krebs, E G; Fischer, E H

    1997-01-01

    The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs. Images PMID:9243503

  6. The activation of p38alpha, and not p38beta, mitogen-activated protein kinase is required for ischemic preconditioning

    PubMed Central

    Sicard, Pierre; Clark, James E.; Jacquet, Sebastien; Mohammadi, Shahrooz; Arthur, J. Simon C.; O'Keefe, Stephen J.; Marber, Michael S.

    2010-01-01

    Numerous studies show that pharmacological inhibition of p38 mitogen-activated protein kinases (p38s) before lethal ischemia prevents conditioning. However, these inhibitors have off-target effects and do not discriminate between the alpha and beta isoforms; the activation of which is thought to have diverse and perhaps opposing actions with p38α aggravating, and p38β reducing, myocardial injury. We adopted a chemical genetic approach using mice in which either the p38α (DRα) or p38β (DRβ) alleles were targeted to substitute the “gatekeeper” threonine residue for methionine, thereby preventing the binding of a pharmacological inhibitor, SB203580. Isolated, perfused wild-type (WT), DRα and DRβ mouse hearts underwent ischemic preconditioning with 4 cycles of 4 min ischemia/6 min reperfusion, with or without SB203580 (10 µM), followed by 30 min of global ischemia and 120 min of reperfusion. In WT and DRβ hearts, SB203580 completely abolished the reduction in myocardial infarction seen with preconditioning and also the phosphorylation of downstream substrates of p38. These effects of SB203580 were not seen in DRα hearts. Furthermore ischemic preconditioning occurred unaltered in p38β null hearts. Contrary to expectation the activation of p38α, and not p38β, is necessary for ischemic preconditioning. Since p38α is also the isoform that leads to lethal myocardial injury, it is unlikely that targeted therapeutic strategies to achieve isoform-selective inhibition will only prevent the harmful consequences of activation. PMID:20188737

  7. Ptc1, a type 2C Ser/Thr phosphatase, inactivates the HOG pathway by dephosphorylating the mitogen-activated protein kinase Hog1.

    PubMed

    Warmka, J; Hanneman, J; Lee, J; Amin, D; Ota, I

    2001-01-01

    The HOG (high-osmolarity glycerol) mitogen-activated protein kinase (MAPK) pathway regulates the osmotic stress response in the yeast Saccharomyces cerevisiae. Three type 2C Ser/Thr phosphatases (PTCs), Ptc1, Ptc2, and Ptc3, have been isolated as negative regulators of this pathway. Previously, multicopy expression of PTC1 and PTC3 was shown to suppress lethality of the sln1Delta strain due to hyperactivation of the HOG pathway. In this work, we show that PTC2 also suppresses sln1Delta lethality. Furthermore, the phosphatase activity of these PTCs was needed for suppression, as mutation of a conserved Asp residue, likely to coordinate a metal ion, inactivated PTCs. Further analysis of Ptc1 function in vivo showed that it inactivates the MAPK, Hog1, but not the MEK, Pbs2. In the wild type, Hog1 kinase activity increased transiently, approximately 12-fold in response to osmotic stress, while overexpression of PTC1 limited activation to approximately 3-fold. In contrast, overexpression of PTC1 did not inhibit phosphorylation of Hog1 Tyr in the phosphorylation lip, suggesting that Ptc1 does not act on Pbs2. Deletion of PTC1 also strongly affected Hog1, leading to high basal Hog1 activity and sustained Hog1 activity in response to osmotic stress, the latter being consistent with a role for Ptc1 in adaptation. In vitro, Ptc1 but not the metal binding site mutant, Ptc1D58N, inactivated Hog1 by dephosphorylating the phosphothreonine but not the phosphotyrosine residue in the phosphorylation lip. Consistent with its role as a negative regulator of Hog1, which accumulates in the nucleus upon activation, Ptc1 was found in both the nucleus and the cytoplasm. Thus, one function of Ptc1 is to inactivate Hog1. PMID:11113180

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

    PubMed

    Ramesh, Geeta; Philipp, Mario T

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

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

    PubMed

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

    2016-09-01

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

  10. Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1.

    PubMed Central

    Hernández, M; Bayón, Y; Sánchez Crespo, M; Nieto, M L

    1997-01-01

    The release of [3H]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A2 (cPLA2) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA2 in SDS/polyacrylamide gels, which is a feature of the activation of cPLA2 by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA2, namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA2 more clearly than the activation of p42-MAK kinase did. Both cPLA2 and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-Jun kinase, also phosphorylated cPLA2 but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA2, and associates the release of arachidonate with nuclear signalling pathways. PMID:9359863

  11. Human Immunodeficiency Virus Type 1 Enters Brain Microvascular Endothelia by Macropinocytosis Dependent on Lipid Rafts and the Mitogen-Activated Protein Kinase Signaling Pathway

    PubMed Central

    Liu, Nancy Q.; Lossinsky, Albert S.; Popik, Waldemar; Li, Xia; Gujuluva, Chandrasekhar; Kriederman, Benjamin; Roberts, Jaclyn; Pushkarsky, Tatania; Bukrinsky, Michael; Witte, Marlys; Weinand, Martin; Fiala, Milan

    2002-01-01

    Brain microvascular endothelial cells (BMVECs) present an incomplete barrier to human immunodeficiency virus type 1 (HIV-1) neuroinvasion. In order to clarify the mechanisms of HIV-1 invasion, we have examined HIV-1 uptake and transcellular penetration in an in vitro BMVEC model. No evidence of productive infection was observed by luciferase, PCR, and reverse transcriptase assays. Approximately 1% of viral RNA and 1% of infectious virus penetrated the BMVEC barrier without disruption of tight junctions. The virus upregulated ICAM-1 on plasma membranes and in cytoplasmic vesiculotubular structures. HIV-1 virions were entangled by microvilli and were taken into cytoplasmic vesicles through surface invaginations without fusion of the virus envelope with the plasma membrane. Subsequently, the cytoplasmic vesicles fused with lysosomes, the virions were lysed, and the vesicles diminished in size. Upon cell entry, HIV-1 colocalized with cholera toxin B, which targets lipid raft-associated GM1 ganglioside. Cholesterol-extracting agents, cyclodextrin and nystatin, and polyanion heparin significantly inhibited virus entry. Anti-CD4 had no effect and the chemokine AOP-RANTES had only a slight inhibitory effect on virus entry. HIV-1 activated the mitogen-activated protein kinase (MAPK) pathway, and inhibition of MAPK/Erk kinase inhibited virus entry. Entry was also blocked by dimethylamiloride, indicating that HIV-1 enters endothelial cells by macropinocytosis. Therefore, HIV-1 penetrates BMVECs in ICAM-1-lined macropinosomes by a mechanism involving lipid rafts, MAPK signaling, and glycosylaminoglycans, while CD4 and chemokine receptors play limited roles in this process. PMID:12050382

  12. Dissecting the role of histidine kinase and HOG1 mitogen-activated protein kinase signalling in stress tolerance and pathogenicity of Parastagonospora nodorum on wheat.

    PubMed

    John, Evan; Lopez-Ruiz, Francisco; Rybak, Kasia; Mousley, Carl J; Oliver, Richard P; Tan, Kar-Chun

    2016-06-01

    The HOG1 mitogen-activated protein kinase (MAPK) pathway is activated through two-component histidine kinase (HK) signalling. This pathway was first characterized in the budding yeast Saccharomyces cerevisiae as a regulator of osmotolerance. The fungus Parastagonospora nodorum is the causal agent of septoria nodorum blotch of wheat. This pathogen uses host-specific effectors in tandem with general pathogenicity mechanisms to carry out its infection process. Genes showing strong sequence homology to S. cerevisiae HOG1 signalling pathway genes have been identified in the genome of P. nodorum. In this study, we examined the role of the pathway in the virulence of P. nodorum on wheat by disrupting putative pathway component genes: HOG1 (SNOG_13296) MAPK and NIK1 (SNOG_11631) hybrid HK. Mutants deleted in NIK1 and HOG1 were insensitive to dicarboximide and phenylpyrrole fungicides, but not a fungicide that targets ergosterol biosynthesis. Furthermore, both Δnik1 and Δhog1 mutants showed increased sensitivity to hyperosmotic stress. However, HOG1, but not NIK1, is required for tolerance to elevated temperatures. HOG1 deletion conferred increased tolerance to 6-methoxy-2-benzoxazolinone, a cereal phytoalexin. This suggests that the HOG1 signalling pathway is not exclusively associated with NIK1. Both Δnik1 and Δhog1 mutants retained the ability to infect and cause necrotic lesions on wheat. However, we observed that the Δhog1 mutation resulted in reduced production of pycnidia, asexual fruiting bodies that facilitate spore dispersal during late infection. Our study demonstrated the overlapping and distinct roles of a HOG1 MAPK and two-component HK signalling in P. nodorum growth and pathogenicity. PMID:26978567

  13. Role of hippocampus mitogen-activated protein kinase phosphatase-1 mRNA expression and DNA methylation in the depression of the rats with chronic unpredicted stress.

    PubMed

    Wang, Chang-Hong; Zhang, Xiao-Li; Li, Yan; Wang, Guo-Dong; Wang, Xin-Kai; Dong, Jiao; Ning, Qiu-Fen

    2015-05-01

    Stressful life events especially the chronic unpredictable stress are the obvious precipitating factors of depression. The biological information transduction in cells plays an important role in the molecular biology mechanism of depression. Mitogen-activated protein kinase phosphatase-1 (MKP-1) regulates the cell physiological activity and involves in the adjustment of neural plasticity, function, and survival. This experiment tried to explore the possible effects of MKP-1 in hippocampus on depression of rats by determining the expression of MKP-1 mRNA and DNA methylation in MKP-1 gene promoter. The animal model was established by chronic unpredictable stress, and evaluated by open-field test and weight changes. All the rats were divided into the sham stimulation, the physiological saline, and the fluoxetine (1.25, 2.50, and 5.00 mg/kg) groups randomly. The expression of MKP-1 mRNA in the hippocampus was measured by RT-PCR and the methylation of MKP-1 promoter DNA was detected by COBRA. The chronic unpredicted stress (1) increased the animal movement scores in open-field test, and fluoxetine could prevent this increasement; (2) increased the body weight, and fluoxetine could not prevent this increasement; and (3) increased MKP-1 mRNA expression in the hippocampus, and fluoxetine could prevent it. However, fluoxetine did not influence the DNA methylation of MKP-1 gene promoter in the hippocampus during the chronic unpredicted stress. MKP-1 in the hippocampus might be involved in the etiology of depression, and DNA methylation of MKP-1 gene promoter in the hippocampus did not related with the depression. PMID:25410305

  14. Neuroprotective effect of osthole against oxygen and glucose deprivation in rat cortical neurons: involvement of mitogen-activated protein kinase pathway.

    PubMed

    Chen, T; Liu, W; Chao, X; Qu, Y; Zhang, L; Luo, P; Xie, K; Huo, J; Fei, Z

    2011-06-01

    Osthole, a bioactive simple coumarin derivative extracted from many medicinal plants such as Cnidium monnieri (L.) Cusson, exerts a broad spectrum of pharmacological activities and is considered to have potential therapeutic applications. The aim of this study was to investigate the potential neuroprotective role of osthole against ischemic injury in vitro, as well as the potential mechanism. Cultured cortical neurons were exposed to oxygen and glucose deprivation (OGD) for 4 h followed by a 24 h reperfusion. Osthole exhibited remarkable neuroprotection in a dose-dependent manner and the effect required presence of osthole during both OGD and reperfusion phases. Western blot was used to examine the activation of three members of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 kinase (p38). We found that osthole prolonged activation of ERK1/2 and prevented activation of JNK. Furthermore, we investigated the effects of MAPKs inhibitors on osthole-induced protection. The results demonstrated that the protection of osthole was partly reversed by PD98059, a selective inhibitor of ERK1/2, but further enhanced by the JNK inhibitor SP600125. In addition, osthole-induced reduction of neuronal apoptosis was abrogated by the ERK1/2 inhibitor PD98059, whereas the total neuronal death was further decreased by the JNK inhibitor SP600125. In summary, these data suggested that osthole had neuroprotective effect against ischemic injury in vitro, and the protection possibly was associated with prolonged activation of ERK1/2 and suppression of JNK activity. PMID:21453755

  15. From pure compounds to complex exposure: Effects of dietary cadmium and lignans on estrogen, epidermal growth factor receptor, and mitogen activated protein kinase signaling in vivo.

    PubMed

    Ali, Imran; Hurmerinta, Teija; Nurmi, Tarja; Berglund, Marika; Rüegg, Joelle; Poutanen, Matti; Halldin, Krister; Mäkelä, Sari; Damdimopoulou, Pauliina

    2016-06-24

    Exposure to environmental endocrine active compounds correlates with altered susceptibility to disease in human populations. Chemical risk assessment is single compound based, although exposure often takes place as heterogeneous mixtures of man-made and natural substances within complex matrices like diet. Here we studied whether the effects of cadmium and enterolactone on endocrine endpoints in dietary exposure can be predicted based on pure compound effects. Ovariectomized estrogen reporter ERE-luciferase (ERE-luc) mice were maintained on diets that intrinsically contain increasing concentrations of cadmium and enterolactone precursors for three and 21 days. The activation of the ERE-luc, epidermal growth factor receptor (EGFR), mitogen activated protein kinase (MAPK)-ERK1/2, and classical estrogen responses were measured. Interactions between the diets and endogenous hormone were evaluated by challenging the animals with 17β-estradiol. Compared to animals on basal purified diet, mice consuming experimental diets were exposed to significantly higher levels of cadmium and enterolactone, yet the exposure remained comparable to typical human dietary intake. Surprisingly, we could not detect effects on endpoints regulated by pure enterolactone, such as ERE-luc activation. However, cadmium accumulation in the liver was accompanied with activation of EGFR and MAPK-ERK1/2 in line with our earlier CdCl2 studies. Further, attenuation of 17β-estradiol-induced ERE-luc response in liver by experimental diets was observed. Our findings indicate that the exposure context can have substantial effects on the activity of endocrine active compounds in vivo. Thus, whenever possible, a context that mimics human exposure should be tested along with pure compounds. PMID:27108949

  16. Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses

    PubMed Central

    Virk, Nasar; Li, Dayong; Tian, Limei; Huang, Lei; Hong, Yongbo; Li, Xiaohui; Zhang, Yafen; Liu, Bo; Zhang, Huijuan; Song, Fengming

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV) while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis. PMID:26222830

  17. Melanin biosynthesis in the maize pathogen Cochliobolus heterostrophus depends on two mitogen-activated protein kinases, Chk1 and Mps1, and the transcription factor Cmr1.

    PubMed

    Eliahu, Noa; Igbaria, Aeid; Rose, Mark S; Horwitz, Benjamin A; Lev, Sophie

    2007-03-01

    The maize pathogen Cochliobolus heterostrophus requires two mitogen-activated protein kinases (MAPKs), Chk1 and Mps1, to produce normal pigmentation. Young colonies of mps1 and chk1 deletion mutants have a white and autolytic appearance, which was partially rescued by a hyperosmotic environment. We isolated the transcription factor Cmr1, an ortholog of Colletotrichum lagenarium Cmr1 and Magnaporthe grisea Pig1, which regulates melanin biosynthesis in C. heterostrophus. Deletion of CMR1 in C. heterostrophus resulted in mutants that lacked dark pigmentation and acquired an orange-pink color. In cmr1 deletion strains the expression of putative scytalone dehydratase (SCD1) and hydroxynaphthalene reductase (BRN1 and BRN2) genes involved in melanin biosynthesis was undetectable, whereas expression of PKS18, encoding a polyketide synthase, was only moderately reduced. In chk1 and mps1 mutants expression of PKS18, SCD1, BRN1, BRN2, and the transcription factor CMR1 itself was very low in young colonies, slightly up-regulated in aging colonies, and significantly induced in hyperosmotic conditions, compared to invariably high expression in the wild type. These findings indicate that two MAPKs, Chk1 and Mps1, affect Cmr1 at the transcriptional level and this influence is partially overridden in stress conditions including aging culture and hyperosmotic environment. Surprisingly, we found that the CMR1 gene was transcribed in both sense and antisense directions, apparently producing mRNA as well as a long noncoding RNA transcript. Expression of the antisense CMR1 was also Chk1 and Mps1 dependent. Analysis of chromosomal location of the melanin biosynthesis genes in C. heterostrophus resulted in identification of a small gene cluster comprising BRN1, CMR1, and PKS18. Since expression of all three genes depends on Chk1 and Mps1 MAPKs, we suggest their possible epigenetic regulation. PMID:17237364

  18. Association of Common Genetic Variants in Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 with Type 2 Diabetes Mellitus in a Chinese Han Population

    PubMed Central

    Li, Ting-Ting; Qiao, Hong; Tong, Hui-Xin; Zhuang, Tian-Wei; Wang, Tong-Tong

    2016-01-01

    Background: A study has identified several novel susceptibility variants of the mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) gene for type 2 diabetes mellitus (T2DM) within the German population. Among the variants, five single nucleotide polymorphisms (SNPs) of MAP4K4 (rs1003376, rs11674694, rs2236935, rs2236936, and rs6543087) showed significant association with T2DM or diabetes-related quantitative traits. We aimed to evaluate whether common SNPs in the MAP4K4 gene were associated with T2DM in the Chinese population. Methods: Five candidate SNPs were genotyped in 996 patients newly diagnosed with T2DM and in 976 control subjects, using the SNPscan™ method. All subjects were recruited from the Second Affiliated Hospital, Harbin Medical University from October 2010 to September 2013. We evaluated the T2DM risk conferred by individual SNPs and haplotypes using logistic analysis, and the association between the five SNPs and metabolic traits in the subgroups. Results: Of the five variants, SNP rs2236935T/C was significantly associated with T2DM in this study population (odds ratio = 1.293; 95% confidence interval: 1.034–1.619, P = 0.025). In addition, among the controls, rs1003376 was significantly associated with an increased body mass index (P = 0.045) and homeostatic model assessment-insulin resistance (P = 0.037). Conclusions: MAP4K4 gene is associated with T2DM in a Chinese Han population, and MAP4K4 gene variants may contribute to the risk toward the development of T2DM. PMID:27174326

  19. Blueberry Opposes β-Amyloid Peptide-Induced Microglial Activation Via Inhibition of p44/42 Mitogen-Activation Protein Kinase

    PubMed Central

    Zhu, Yuyan; Bickford, Paula C.; Sanberg, Paul; Giunta, Brian

    2008-01-01

    Abstract Alzheimer's Disease (AD) is the most common age-related dementia, with a current prevalence in excess of five million individuals in the United States. The aggregation of amyloid-beta (Aβ) into fibrillar amyloid plaques is a key pathological event in the development of the disease. Microglial proinflammatory activation is widely known to cause neuronal and synaptic damage that correlates with cognitive impairment in AD. However, current pharmacological attempts at reducing neuroinflammation mediated via microglial activation have been largely negative in terms of slowing AD progression. Previously, we have shown that microglia express proinflammatory cytokines and a reduced capacity to phagocytose Aβ in the context of CD40, Aβ peptides and/or lipopolysaccharide (LPS) stimulation, a phenomenon that can be opposed by attenuation of p44/42 mitogen-activated protein kinase (MAPK) signaling. Other groups have found that blueberry (BB) extract both inhibits phosphorylation of this MAPK module and also improves cognitive deficits in AD model mice. Given these considerations and the lack of reduced Aβ quantities in behaviorally improved BB-fed mice, we wished to determine whether BB supplementation would alter the microglial proinflammatory activation state in response to Aβ. We found that BB significantly enhances microglial clearance of Aβ, inhibits aggregation of Aβ1–42, and suppresses microglial activation, all via suppression of the p44/42 MAPK module. Thus, these data may explain the previously observed behavioral recovery in PSAPP mice and suggest a means by which dietary supplementation could mitigate an undesirable microglial response toward fibrillar Aβ. PMID:18789000

  20. Targeting mitogen-activated protein kinase kinase with the inhibitor PD0325901 decreases hepatocellular carcinoma growth in vitro and in mouse model systems.

    PubMed

    Hennig, Matthew; Yip-Schneider, Michele T; Wentz, Sabrina; Wu, Huangbing; Hekmatyar, S K; Klein, Patrick; Bansal, Navin; Schmidt, C Max

    2010-04-01

    Hepatocellular carcinoma (HCC) is a common cause of death from solid organ malignancy worldwide. Extracellular signal-regulated/mitogen-activated protein kinase kinase (MEK) signaling is a critical growth regulatory pathway in HCC. Targeting MEK with a novel small molecule inhibitor, PD0325901, may inhibit HCC tumorigenesis. PD0325901 (0.01-100 nM) inhibited growth and MEK activity in vitro in immortalized murine transforming growth factor alpha (TGF-alpha) transgenic hepatocyte (TAMH) cells, derived from the livers of TGF-alpha transgenic mice. Treatment of athymic mice bearing TAMH flank tumors with vehicle or PD0325901 (20 mg/kg) revealed a significant reduction of MEK activity ex vivo 24 hours after a single PD0325901 dose. The growth rate of TAMH flank tumors over 16 days was reduced threefold in the treatment arm (1113 +/- 269% versus 3077 +/- 483%, P < 0.01). PD0325901 exhibited similar inhibitory effects in HepG2 and Hep3B human HCC cells in vitro and in Hep3B flank tumors in vivo. To confirm this in a developmental model, MT-42 (CD-1) TGF-alpha mice were treated with vehicle or PD0325901 (20 mg/kg) for 5 weeks. Gross HCC was detected in 47% and 13.3% of the control and treatment mice, respectively. Tumor growth suppression by PD0325901 relative to vehicle was also shown by magnetic resonance imaging. These studies provide compelling preclinical evidence that targeting MEK in human clinical trials may be promising for the treatment of HCC. PMID:20112426

  1. Isoquercitrin inhibits the progression of pancreatic cancer in vivo and in vitro by regulating opioid receptors and the mitogen-activated protein kinase signalling pathway.

    PubMed

    Chen, Quan; Li, Ping; Li, Ping; Xu, Yong; Li, Yang; Tang, Bo

    2015-02-01

    of the mitogen-activated protein kinase (MAPK) signalling pathway. PMID:25434366

  2. Targeting p38 mitogen-activated protein kinase signaling restores subventricular zone neural stem cells and corrects neuromotor deficits in Atm knockout mouse.

    PubMed

    Kim, Jeesun; Wong, Paul K Y

    2012-07-01

    Ataxia-telangiectasia (A-T) is a progressive degenerative disorder that results in major neurological disability. In A-T patients, necropsy has revealed atrophy of cerebellar cortical layers along with Purkinje and granular cell loss. We have previously identified an oxidative stress-mediated increase in phospho-p38 mitogen-activated protein kinase (MAPK) and the resultant downregulation of Bmi-1 and upregulation of p21 as key components of the mechanism causing defective proliferation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of Atm(-/-) mice. However, the in vivo aspect of alteration in SVZ tissue and the functional significance of p38MAPK activation in NSCs for neuropathogenesis of ATM deficiency remain unknown. Here we show that the NSC population was abnormally decreased in the SVZ of 3-month-old Atm(-/-) mice; this decrease was accompanied by p38MAPK activation. However, after a 2-month treatment with the p38MAPK inhibitor SB203580, starting at 1 month old, Atm(-/-) mice showed restoration of normal levels of Bmi-1 and p21 with the rescue of NSC population in the SVZ. In addition, treated Atm(-/-) mice exhibited more Purkinje cells in the cerebellum. Most importantly, motor coordination of Atm(-/-) mice was significantly improved in the treatment group. Our results show for the first time in vivo evidence of depleted NSCs in the SVZ of Atm(-/-) mice and also demonstrate that pharmacologic inhibition of p38MAPK signaling has the potential to treat neurological defects of A-T. This study provides a promising approach targeting the oxidative stress-dependent p38 signaling pathway not only for A-T but also for other neurodegenerative disorders. PMID:23197859

  3. Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

    PubMed

    Virk, Nasar; Li, Dayong; Tian, Limei; Huang, Lei; Hong, Yongbo; Li, Xiaohui; Zhang, Yafen; Liu, Bo; Zhang, Huijuan; Song, Fengming

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV) while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis. PMID:26222830

  4. Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

    PubMed

    Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

    2016-06-01

    We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. PMID:27161454

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    2016-08-26

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

  7. The SrkA Kinase Is Part of the SakA Mitogen-Activated Protein Kinase Interactome and Regulates Stress Responses and Development in Aspergillus nidulans

    PubMed Central

    Jaimes-Arroyo, Rafael; Lara-Rojas, Fernando; Bayram, Özgür; Valerius, Oliver; Braus, Gerhard H.

    2015-01-01

    Fungi and many other eukaryotes use specialized mitogen-activated protein kinases (MAPK) of the Hog1/p38 family to transduce environmental stress signals. In Aspergillus nidulans, the MAPK SakA and the transcription factor AtfA are components of a central multiple stress-signaling pathway that also regulates development. Here we characterize SrkA, a putative MAPK-activated protein kinase, as a novel component of this pathway. ΔsrkA and ΔsakA mutants share a derepressed sexual development phenotype. However, ΔsrkA mutants are not sensitive to oxidative stress, and in fact, srkA inactivation partially suppresses the sensitivity of ΔsakA mutant conidia to H2O2, tert-butyl-hydroperoxide (t-BOOH), and menadione. In the absence of stress, SrkA shows physical interaction with nonphosphorylated SakA in the cytosol. We show that H2O2 induces a drastic change in mitochondrial morphology consistent with a fission process and the relocalization of SrkA to nuclei and mitochondria, depending on the presence of SakA. SakA-SrkA nuclear interaction is also observed during normal asexual development in dormant spores. Using SakA and SrkA S-tag pulldown and purification studies coupled to mass spectrometry, we found that SakA interacts with SrkA, the stress MAPK MpkC, the PPT1-type phosphatase AN6892, and other proteins involved in cell cycle regulation, DNA damage response, mRNA stability and protein synthesis, mitochondrial function, and other stress-related responses. We propose that oxidative stress induces DNA damage and mitochondrial fission and that SakA and SrkA mediate cell cycle arrest and regulate mitochondrial function during stress. Our results provide new insights into the mechanisms by which SakA and SrkA regulate the remodelling of cell physiology during oxidative stress and development. PMID:25820520

  8. Natural feed contaminant zearalenone decreases the expressions of important pro- and anti-inflammatory mediators and mitogen-activated protein kinase/NF-κB signalling molecules in pigs.

    PubMed

    Pistol, Gina Cecilia; Gras, Mihail Alexandru; Marin, Daniela Eliza; Israel-Roming, Florentina; Stancu, Mariana; Taranu, Ionelia

    2014-02-01

    Zearalenone (ZEA) is an oestrogenic mycotoxin produced by Fusarium species, considered to be a risk factor from both public health and agricultural perspectives. In the present in vivo study, a feeding trial was conducted to evaluate the in vivo effect of a ZEA-contaminated diet on immune response in young pigs. The effect of ZEA on pro-inflammatory (TNF-α, IL-8, IL-6, IL-1β and interferon-γ) and anti-inflammatory (IL-10 and IL-4) cytokines and other molecules involved in inflammatory processes (matrix metalloproteinases (MMP)/tissue inhibitors of matrix metalloproteinases (TIMP), nuclear receptors: PPARγ and NF-κB1, mitogen-activated protein kinases (MAPK): mitogen-activated protein kinase kinase kinase 7 (TAK1)/mitogen-activated protein kinase 14 (p38α)/mitogen-activated protein kinase 8 (JNK1)/ mitogen-activated protein kinase 9 (JNK2)) in the liver of piglets was investigated. The present results showed that a concentration of 316 parts per billion ZEA leads to a significant decrease in the levels of pro- and anti-inflammatory cytokines at both gene expression and protein levels, correlated with a decrease in the levels of other inflammatory mediators, MMP and TIMP. The results also showed that dietary ZEA induces a dramatic reduction in the expressions of NF-κB1 and TAK1/p38α MAPK genes in the liver of the experimentally intoxicated piglets, and has no effect on the expression of PPARγ mRNA. The present results suggest that the toxic action of ZEA begins in the upstream of the MAPK signalling pathway by the inhibition of TAK1, a MAPK/NF-κB activator. In conclusion, the present study shows that ZEA alters several important parameters of the hepatic cellular immune response. From an economic point of view, these data suggest that, in pigs, ZEA is not only a powerful oestrogenic mycotoxin but also a potential hepatotoxin when administered through the oral route. Therefore, the present results represent additional data from cellular and molecular levels

  9. 2,2',4,4'-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-{kappa}B

    SciTech Connect

    Bezdecny, Steven A.; Karmaus, Peer; Roth, Robert A.; Ganey, Patricia E. . E-mail: ganey@msu.edu

    2007-06-15

    Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2',4,4'-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A{sub 2} with subsequent release of arachidonic acid (AA) and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 min. The increase in COX-2 mRNA was associated with release of {sup 3}H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-{kappa}B and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A{sub 2}, reduced {sup 3}H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter {sup 3}H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-{kappa}B. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-{kappa}B prevented the 2244-TCB

  10. Synergistic effects of p38 mitogen-activated protein kinase inhibition with a corticosteroid in alveolar macrophages from patients with chronic obstructive pulmonary disease.

    PubMed

    Armstrong, J; Harbron, C; Lea, S; Booth, G; Cadden, P; Wreggett, K A; Singh, D

    2011-09-01

    Corticosteroids partially suppress cytokine production by chronic obstructive pulmonary disease (COPD) alveolar macrophages. p38 mitogen-activated protein kinase (MAPK) inhibitors are a novel class of anti-inflammatory drug. We have studied the effects of combined treatment with a corticosteroid and a p38 MAPK inhibitor on cytokine production by COPD alveolar macrophages, with the aim of investigating dose-sparing and efficacy-enhancing effects. Alveolar macrophages from 10 patients with COPD, six smokers, and six nonsmokers were stimulated with lipopolysaccharide (LPS) after preincubation with five concentrations of dexamethasone alone, five concentrations of the p38 MAPK inhibitor 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB-796) alone, and all combinations of these concentrations. After 24 h, the supernatants were analyzed for interleukin (IL)-8, IL-6, tumor necrosis factor α (TNFα), granulocyte macrophage-colony-stimulating factor (GM-CSF), IL-1α, IL-1β, IL-1ra, IL-10, monocyte chemoattractant protein 3, macrophage-derived chemokine (MDC), and regulated on activation normal T cell expressed and secreted (RANTES). The effect of dexamethasone on p38 MAPK activation was analyzed by Western blotting. Dexamethasone and BIRB-796 both reduced LPS-induced cytokine production in a dose-dependent manner in all subject groups, with no differences between groups. Increasing the concentration of BIRB-796 in combination with dexamethasone produced progressively greater inhibition of cytokine production than dexamethasone alone. There were significant efficacy-enhancing benefits and synergistic dose-sparing effects (p < 0.05) for the combination treatment for IL-8, IL-6, TNFα, GM-CSF, IL-1ra, IL-10, MDC, and RANTES in one or more subject groups. Dexamethasone had no effect on LPS-induced p38 MAPK activation. We conclude that p38 MAPK activation in alveolar macrophages is corticosteroid-insensitive. Combining a p38

  11. Mitogen-activated protein kinases and nuclear factor-kappaB regulate Helicobacter pylori-mediated interleukin-8 release from macrophages.

    PubMed Central

    Bhattacharyya, Asima; Pathak, Shresh; Datta, Simanti; Chattopadhyay, Santanu; Basu, Joyoti; Kundu, Manikuntala

    2002-01-01

    Gastric infection, as well as inflammation, caused by Helicobacter pylori, activates the production of cytokines and chemokines by mononuclear cells; interleukin-8 (IL-8) is one of the major inflammatory chemokines. Since H. pylori does not invade mucosal tissue, we observed the effect of the water extract of H. pylori (HPE), containing shed factors, on the production of IL-8 by human peripheral blood monocytes and the human monocyte cell line THP-1. HPE-treatment induced activation of the mitogen-activated protein kinases (MAPKs) ERK (extracellular signal-regulated kinase), p38 and JNK (c-Jun N-terminal kinase), an effect which was not dependent on the presence of the cag pathogenicity island. p38 MAPK activation was sustained. The specific inhibitors, U0126 (for ERK1/2 signalling) and SB203580 (for p38 MAPK signalling), both abrogated IL-8 secretion from HPE-treated THP-1. Dominant-negative mutants of the upstream kinases MEK1 (MAPK/ERK kinase 1), MKK (MAPK kinase) 6 and MKK7 also inhibited IL-8 secretion, pointing to a role of all three MAPKs in HPE-mediated IL-8 release. The inhibitory effects of polymyxin B and anti-CD14 antibody suggested that the effect of HPE on MAPKs was mediated by H. pylori lipopolysaccharide (LPS). By analysis of IL-8-promoter-driven luciferase gene expression, we observed that the effects of HPE-induced nuclear factor-kappaB (NF-kappaB) activation and MAPK signalling were mediated at the level of the IL-8 promoter. While ERK1/2 activation could be linked to enhanced DNA binding of activator protein-1 (AP-1), p38 MAPK signalling did not affect AP-1 DNA binding. Taken together, these results provide the first evidence that LPS from H. pylori stimulates IL-8 release from cells of the monocytic lineage through activation of NF-kappaB and signalling along MAPK cascades. The stimulation of MAPK signalling in macrophages by LPS of H. pylori amplifies the inflammatory response associated with gastric H. pylori infection and needs to be taken

  12. N-Farnesyloxy-norcantharimide inhibits progression of human leukemic Jurkat T cells through regulation of mitogen-activated protein kinase and interleukin-2 production

    PubMed Central

    Chang, Ming-Che; Wu, Jin-Yi; Liao, Hui-Fen; Chen, Yu-Jen

    2015-01-01

    This study investigated the anticancer effects of N-farnesyloxy-norcantharimide (NOC15), a newly synthesized norcantharidin (NCTD) analogue, on human leukemic Jurkat T cells and the signaling pathway underlying its effects. We found that the half maximal inhibitory concentration (IC50) of NOC15 on Jurkat T cells is 1.4 μmol/l, which is 11.14-fold (=15.6÷1.4) smaller than the 15.6 μmol/l of NCTD on Jurkat T cells, whereas the IC50 of NOC15 on human normal lymphoblast (HNL) is 207.9 μmol/l, which is 8.17-fold (=1698.0÷207.8) smaller than the 1698.0 μmol/l of NCTD on HNL cells. These results indicated that NOC15 exerts a higher anticancer effect on Jurkat T cells and has higher toxicity toward HNL cells than NCTD. Thus, NOC15 is 1.36-fold (=11.14÷8.17) beneficial as an anticancer agent toward Jurkat T cells compared with NCTD. Moreover, NOC15 can increase the percentage of cells in the sub-G1 phase and reduce the cell viability of Jurkat T cells, stimulate p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) of mitogen-activated protein kinases (MAPKs) signaling pathway, and inhibit calcineurin expression and interleukin-2 (IL-2) production. However, NOC15 exerted no effects on the Jun-N-terminal kinase 1/2 (JNK1/2) signaling pathway, the production of IL-8, and tumor necrosis factor-α. We conclude that the anticancer activity of the newly synthesized NOC15 is 1.36-fold beneficial than NCTD as an anticancer agent and that NOC15 can increase the percentage of cells in the sub-G1 phase through the stimulation of p38 and ERK1/2 of the MAPK signaling pathway and the inhibition of calcineurin expression and IL-2 production. The NOC15 may have the potential of being developed into an anticancer agent in the future. PMID:26288134

  13. 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.

    PubMed Central

    von Manteuffel, S R; Gingras, A C; Ming, X F; Sonenberg, N; Thomas, G

    1996-01-01

    It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However, the immunosuppressant rapamycin blocks serum-induced 4E-BP1 phosphorylation and, in parallel, p70s6k activation, with no apparent effect on p42mapk activation. Consistent with this finding, the kinetics of serum-induced 4E-BP1 phosphorylation closely follow those of p70s6k activation rather than those of p42mapk. More striking, insulin, which does not induce p42mapk activation in human 293 cells or Swiss mouse 3T3 cells, induces 4E-BP1 phosphorylation and p70s6k activation in both cell types. Anisomycin, which, like insulin, does not activate p42mapk, promotes a small parallel increase in 4E-BP1 phosphorylation and p70s6k activation. The insulin effect on 4E-BP1 phosphorylation and p70s6k activation in both cell types is blocked by SQ20006, wortmannin, and rapamycin. These three inhibitors have no effect on p42mapk activation induced by phorbol 12-tetradecanoate 13-acetate, though wortmannin partially suppresses both the p70s6k response and the 4E-BP1 response. Finally, in porcine aortic endothelial cells stably transfected with either the wild-type platelet-derived growth factor receptor or a mutant receptor bearing the double point mutation 740F/751F, p42mapk activation in response to platelet-derived growth factor is unimpaired, but increased 4E-BP1 phosphorylation is ablated, as previously reported for p70s6k. The data presented here demonstrate that 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8633019

  14. Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

    PubMed Central

    Dickenson, John M; Blank, Jonathan L; Hill, Stephen J

    1998-01-01

    The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric Gi/Go protein-coupled and Gq/G11 protein-coupled receptors. The aims of the current study were: (i) to investigate whether the Gi/Go protein-coupled adenosine A1 receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block Gi/Go-dependent pathways).Adenosine A1 receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).Activation of the endogenous P2Y2 purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both Gi/Go protein and Gq protein-dependent pathways in the overall response to UTP.CPA and UTP stimulated concentration

  15. Epidermal growth factor-mediated mitogen-activated protein kinase3/1 pathway is conducive to in vitro maturation of sheep oocytes.

    PubMed

    Ni, Hemin; Sheng, Xihui; Cui, Xu; Gu, Meichao; Liu, Yunhai; Qi, Xiaolong; Xing, Shuhan; Guo, Yong

    2015-01-01

    Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo's capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10⁻⁴ mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10-6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10⁻⁴ mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10⁻⁶ mol/l U0126 group and 42.6% in 10⁻⁴ mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10⁻⁴ mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes. PMID:25799554

  16. Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

    SciTech Connect

    Muchir, Antoine; Wu, Wei; Sera, Fusako; Homma, Shunichi; Worman, Howard J.

    2014-10-03

    Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left

  17. The Role of Mitogen-Activated Protein (MAP) Kinase Signaling Components in the Fungal Development, Stress Response and Virulence of the Fungal Cereal Pathogen Bipolaris sorokiniana

    PubMed Central

    Leng, Yueqiang; Zhong, Shaobin

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) have been demonstrated to be involved in fungal development, sexual reproduction, pathogenicity and/or virulence in many filamentous plant pathogenic fungi, but genes for MAPKs in the fungal cereal pathogen Bipolaris sorokiniana have not been characterized. In this study, orthologues of three MAPK genes (CsSLT2, CsHOG1 and CsFUS3) and one MAPK kinase kinase (MAPKKK) gene (CsSTE11) were identified in the whole genome sequence of the B. sorokiniana isolate ND90Pr, and knockout mutants were generated for each of them. The ∆Csfus3 and ∆Csste11 mutants were defective in conidiation and formation of appressoria-like structures, showed hypersensitivity to oxidative stress and lost pathogenicity on non-wounded leaves of barley cv. Bowman. When inoculated on wounded leaves of Bowman, the ∆Csfus3 and ∆Csste11 mutants were reduced in virulence compared to the wild type. No morphological changes were observed in the ∆Cshog1 mutants in comparison with the wild type; however, they were slightly reduced in growth under oxidative stress and were hypersensitive to hyperosmotic stress. The ∆Cshog1 mutants formed normal appressoria-like structures but were reduced in virulence when inoculated on Bowman leaves. The ∆Csslt2 mutants produced more vegetative hyphae, had lighter pigmentation, were more sensitive to cell wall degrading enzymes, and were reduced in virulence on Bowman leaves, although they formed normal appressoria like the wild type. Root infection assays indicated that the ∆Cshog1 and ∆Csslt2 mutants were able to infect barley roots while the ∆Csfus3 and ∆Csste11 failed to cause any symptoms. However, no significant difference in virulence was observed for ∆Cshog1 mutants while ∆Csslt2 mutants showed significantly reduced virulence on barley roots in comparison with the wild type. Our results indicated that all of these MAPK and MAPKKK genes are involved in the regulation of fungal development under

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

    PubMed

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

    2016-05-01

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

  19. Sensitization of apoptotically-resistant breast carcinoma cells to TNF and TRAIL by inhibition of p38 mitogen-activated protein kinase signaling.

    PubMed

    Weldon, Christopher B; Parker, Amanda P; Patten, Daniel; Elliott, Steven; Tang, Yan; Frigo, Daniel E; Dugan, Christine M; Coakley, Erin L; Butler, Nancy N; Clayton, John L; Alam, Jawed; Curiel, Tyler J; Beckman, Barbara S; Jaffe, Bernard M; Burow, Matthew E

    2004-06-01

    The mitogen-activated protein kinase (MAPK) cascade is a critical component in the regulation of cell survival and proliferation decisions. In breast carcinoma cells, activation of the p38-MAPK member of this family occurs in response to pro-inflammatory cytokines and cellular stress. The involvement of p38-MAPK in the activation of the transcription factor, NF-kappaB, suggests a potential role and mechanism for regulation of cell survival and drug resistance. Generation of the resistant MCF-7 variant (MCF-7TN-R) was achieved by prolonged exposure of MCF-7N cells to increasing concentrations of TNF. Differences in MAPK activation and function in the MCF-7 cell variants were determined. The role of the p38-MAPK pathway in regulation of resistance was determined using pharmacological (SB 203580) or molecular [Dominant Inhibitory (DI)-p38] inhibition. The effect of p38 inhibition on NF-kappaB transcriptional activation was analyzed. As compared to the sensitive MCF-7N parent cell line, the MCF-7TN-R cell line displayed significant resistance to TNF- and TRAIL-induced cell death. Analysis of the expression and phosphorylation of members of the MAPK family revealed an increased basal activation of p38 in the MCF-7TN-R variant. The p38-mediated phosphorylation and transcriptional activity were suppressed by pharmacologic inhibition with SB 230580. Treatment of MCF-7TN-R cells with SB partially restored sensitivity to TNF-induced cell death. In addition, use of a DI-p38 construct with or without the addition to TNF induced cell death, thus restoring TNF-sensitivity to these cells. The ability of p38 inhibition to restore apoptotic sensitivity was correlated with suppression of the TNF-induced cell survival pathway, NF-kappaB. The increased activation of p38-MAPK in MCF-7TN-R cells demonstrates that this signaling pathway through activation of NF-kappaB is an important route for control of resistance to cell death in breast carcinoma. Molecular and pharmacological

  20. Epidermal Growth Factor-Mediated Mitogen-Activated Protein Kinase3/1 Pathway Is Conducive to In Vitro Maturation of Sheep Oocytes

    PubMed Central

    Cui, Xu; Gu, Meichao; Liu, Yunhai; Qi, Xiaolong; Xing, Shuhan; Guo, Yong

    2015-01-01

    Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo’s capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10−4 mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10−6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10−4 mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10−6 mol/l U0126 group and 42.6% in 10−4 mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10−4 mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes. PMID:25799554

  1. Mitogenic activity of edible mushroom lectins.

    PubMed

    Ho, J C K; Sze, S C W; Shen, W Z; Liu, W K

    2004-03-17

    A special group of lectins were isolated from three popular Asian edible mushrooms: Volvariella volvacea, Pleurotus flabellatus and Hericium erinacium, and their mitogenic activities towards mouse T cells were compared to the extensively investigated Agaricus bisporus lectin (ABL) and the Jack bean lectin, Concanavalin A (Con A). Among the four mushroom lectins tested, V. volvacea lectin (VVL) exhibited strong mitogenic activity as demonstrated by 3H-thymidine incorporation, which was at least 10-fold more effective than that of Con A, and the other mushroom lectins did not exhibit any proliferative activity. Treatment with VVL and ABL resulted in activation of the protein tyrosine kinase, p56lck, and expression of early activation markers, CD69 and CD25, but only VVL induced intracellular calcium influx while ABL triggered cell death. The calcium influx was sensitive to calcium channel antagonists such as nifedipine and verapamil. The P. flabellatus lectin (PFL) and H. erinacium lectin (HEL) did not stimulate p56lck expression and cell proliferation. Neither of these lectins interfered with Con A-mediated lymphocyte proliferation, which further indicated that both PFL and HEL were non-mitogenic. Taken all results together, VVL induced mitogenesis through T cell receptors and the subsequent calcium signaling pathway. PMID:15026140

  2. Activation of mitogen-activated protein kinases and AP-1 transcription factor in ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats.

    PubMed

    Hu, Xiaoming; Flaws, Jodi A; Sipes, I Glenn; Hoyer, Patricia B

    2002-09-01

    Previous studies have demonstrated that ovotoxicity induced in small preantral (primordial and primary) ovarian follicles by 4-vinylcyclohexene diepoxide (VCD) in rats is likely via acceleration of the normal process of atresia (apoptosis). This acceleration is associated with increased activities of caspase cascades, changes in subcellular distribution of Bcl-2 family members, and alteration of estrogen receptor-mediated signaling pathways. The present study was designed to investigate possible effects of VCD dosing on the mitogen-activated protein kinases (MAPK)/AP-1 signaling pathways in rat ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg i.p., 1 day--a time when ovotoxicity has not been initiated) or dosed daily for 10 or 15 days (80 mg/kg i.p.; 10 days--a time when the earliest signs of impending follicular destruction is seen, 15 days--a time when significant ovotoxicity is underway). Four hours following the final dose, ovaries and livers were collected. Ovarian small (25-100 microm) and large (100-250 microm) preantral follicles were isolated, and cytosolic or nuclear extracts were prepared from follicles and livers for analyses. Activities of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal protein kinase (JNK), and p38 kinase, were determined in follicular and liver cytosolic extracts, and AP-1 DNA binding activity was determined in follicular and liver nuclear extracts. Compared with control, a single dose of VCD caused a decrease in JNK activity and an increase of AP-1 binding activity in isolated small ovarian follicles. After repeated daily dosing with VCD for 10 or 15 days, JNK and p38 kinase activities in small ovarian follicles were increased (p38 kinase: 1.64 +/- 0.14 for 10 days, 1.48 +/- 0.11 for 15 days, VCD/control, P < 0.01; JNK: 1.44 +/- 0.11 for 10 days, 1.37 +/- 0.06 for 15 days, VCD/control, P < 0.01) and AP-1 binding activity in small ovarian follicles was decreased (10 days, 0

  3. Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle. Role of endothelial differentiation gene 1, c-Src tyrosine kinase and phosphoinositide 3-kinase.

    PubMed Central

    Rakhit, S; Conway, A M; Tate, R; Bower, T; Pyne, N J; Pyne, S

    1999-01-01

    We report here that cultured airway smooth muscle cells contain transcripts of endothelial differentiation gene 1 (EDG-1), a prototypical orphan Gi-coupled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PTX)-sensitive manner in these cells. An essential role for c-Src was confirmed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhibitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An essential role for PI-3K was supported by experiments that showed that PI-3K activity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatment of cells with PTX. Finally, we have shown that the co-stimulation of cells with platelet-derived growth factor (PDGF) and S1P (which failed to stimulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 min stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demonstrate that S1P activates c-Src and Grb-2-PI-3K (intermediates in the p42/p44 MAPK cascade) via a PTX-sensitive mechanism. This action of S1P is consistent with the stimulation of EDG-1 receptors. S1P might also function as a co-mitogen with PDGF, producing a more robust activation of a common permissive signal transduction pathway linked to DNA synthesis. PMID:10051434

  4. Role of Mitogen-Activated Protein Kinases in Peptidoglycan-Induced Expression of Inducible Nitric Oxide Synthase and Nitric Oxide in Mouse Peritoneal Macrophages: Extracellular Signal-Related Kinase, a Negative Regulator ▿ †

    PubMed Central

    Bhatt, Kunal H.; Sodhi, Ajit; Chakraborty, Rituparna

    2011-01-01

    The expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) are important host defense mechanisms against pathogens in mononuclear phagocytes. The objectives of this study were to examine the roles of mitogen-activated protein kinases (MAPKs) and transcription factors (nuclear factor-κB [NF-κB] and activating protein 1 [AP-1]) in peptidoglycan (PGN)-induced iNOS expression and NO production in macrophages. PGN is a cell wall component of Gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo. PGN stimulates the activation of all three classes of MAPKs, extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38mapk in macrophages, albeit with differential activation kinetics. Using a selective inhibitor of JNK (SP600125) and JNK1/2 small interfering RNA (siRNA) knocked-down macrophages, it was observed that PGN-induced iNOS and NO expression is significantly inhibited. This suggested that JNK MAPK plays an essential role in PGN-induced iNOS expression and NO production. In contrast, inhibition of the ERK pathway using PD98059 dose dependently enhanced PGN-induced iNOS expression and NO production. PGN-induced ERK activation was attenuated in ERK1/2 siRNA knocked-down macrophages; however, NO and iNOS expression were significantly enhanced. An electrophoretic mobility shift assay showed that SP600125 inhibited PGN-induced NF-κB and AP-1 activation, whereas inhibition of the ERK pathway enhanced NF-κB activation, but with no effect on AP-1. These results indicate that the JNK MAPK positively regulate PGN-induced iNOS and NO expression by activating NF-κB and AP-1 transcription factors, whereas the ERK pathway plays a negative regulatory role via affecting NF-κB activity. PMID:21450974

  5. Hepcidin promotes osteogenic differentiation through the bone morphogenetic protein 2/small mothers against decapentaplegic and mitogen-activated protein kinase/P38 signaling pathways in mesenchymal stem cells

    PubMed Central

    LU, HUADING; LIAN, LIYI; SHI, DEHAI; ZHAO, HUIQING; DAI, YUHU

    2015-01-01

    The ability of mesenchymal stem cells (MSCs) to differentiate into osteogenic lineages requires management for their future use in treating bone destruction and osteoporosis. Hepcidin is closely associated with bone metabolism, however, it remains to be elucidated whether hepcidin affects osteogenic differentiation in MSCs. The present study demonstrated that hepcidin enhanced osteoblastic differentiation and mineralization, which was manifested by an upregulation in the differentiation markers alkaline phosphatase and osteogenic genes. Furthermore, the expression levels of bone morphogenetic proteins and small mothers against decapentaplegic homologs were concomitantly increased following hepcidin treatment. In addition, the p38 mitogen-activated protein kinase may be an upstream kinase for osteoblastic differentiation. Thus, hepcidin may be important in the osteogenic differentiation of MSCs and may be considered as a target in the development of therapies for pathological bone loss. PMID:25351366

  6. The Heat Shock Factor A4A Confers Salt Tolerance and Is Regulated by Oxidative Stress and the Mitogen-Activated Protein Kinases MPK3 and MPK61[C][W][OPEN

    PubMed Central

    Pérez-Salamó, Imma; Papdi, Csaba; Rigó, Gábor; Zsigmond, Laura; Vilela, Belmiro; Lumbreras, Victoria; Nagy, István; Horváth, Balázs; Domoki, Mónika; Darula, Zsuzsa; Medzihradszky, Katalin; Bögre, László; Koncz, Csaba; Szabados, László

    2014-01-01

    Heat shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here, we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis (Arabidopsis thaliana). Estradiol induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast (Saccharomyces cerevisiae) two-hybrid and bimolecular fluorescence complementation assays, HSFA4A shows homomeric interaction, which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, serine-309 being the major phosphorylation site. Activation of the MPK3 and MPK6 mitogen-activated protein kinase pathway led to the transcriptional activation of the HEAT SHOCK PROTEIN17.6A gene. In agreement that mutation of serine-309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HEAT SHOCK PROTEIN17.6A. These data suggest that HSFA4A is a substrate of the MPK3/MPK6 signaling and that it regulates stress responses in Arabidopsis. PMID:24676858

  7. Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway*

    PubMed Central

    Cohen, Adiel; Kupiec, Martin; Weisman, Ronit

    2014-01-01

    The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling. PMID:24928510

  8. Anti-inflammatory effects of cordycepin in lipopolysaccharide-stimulated RAW 264.7 macrophages through Toll-like receptor 4-mediated suppression of mitogen-activated protein kinases and NF-κB signaling pathways

    PubMed Central

    Choi, Yung Hyun; Kim, Gi-Young; Lee, Hye Hyeon

    2014-01-01

    Cordycepin is the main functional component of the Cordyceps species, which has been widely used in traditional Oriental medicine. This compound possesses many pharmacological properties, such as an ability to enhance immune function, as well as antioxidant, antiaging, and anticancer effects. In the present study, we investigated the anti-inflammatory effects of cordycepin using a murine macrophage RAW 264.7 cell model. Our data demonstrated that cordycepin suppressed production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 by inhibiting inducible NO synthase and cyclooxygenase-2 gene expression. Cordycepin also inhibited the release of proinflammatory cytokines, including tumor necrosis factor-alpha and interleukin-1-beta, through downregulation of respective mRNA expression. In addition, pretreatment with cordycepin significantly inhibited lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activating protein kinases and attenuated nuclear translocation of NF-κB by LPS, which was associated with abrogation of inhibitor kappa B-alpha degradation. Furthermore, cordycepin potently inhibited the binding of LPS to macrophages and LPS-induced Toll-like receptor 4 and myeloid differentiation factor 88 expression. Taken together, the results suggest that the inhibitory effects of cordycepin on LPS-stimulated inflammatory responses in RAW 264.7 macrophages are associated with suppression of mitogen-activating protein kinases and activation of NF-κB by inhibition of the Toll-like receptor 4 signaling pathway. PMID:25342887

  9. Genome-wide identification and expression analysis of the mitogen-activated protein kinase gene family from banana suggest involvement of specific members in different stages of fruit ripening.

    PubMed

    Asif, Mehar Hasan; Lakhwani, Deepika; Pathak, Sumya; Bhambhani, Sweta; Bag, Sumit K; Trivedi, Prabodh Kumar

    2014-03-01

    Mitogen-activated protein kinases (MAPKs) are important components of the tripartite mitogen-activated protein kinase signaling cascade and play an important role in plant growth and development. Although members of the MAPK gene family have been identified in model plants, little information is available regarding this gene family in fruit crops. In this study, we carried out a computational analysis using the Musa Genome database to identify members of the MAPK gene family in banana, an economically important crop and the most popular fruit worldwide. Our analysis identified 25 members of the MAP kinase (MAPK or MPK) gene family. Phylogenetic analyses of MPKs in Arabidopsis, Oryza, and Populus have classified these MPKs into four subgroups. The presence of conserved domains in the deduced amino acid sequences, phylogeny, and genomic organization strongly support their identity as members of the MPK gene family. Expression analysis during ethylene-induced banana fruit ripening suggests the involvement of several MPKs in the ethylene signal transduction pathway that are necessary for banana fruit ripening. Analysis of the cis-regulatory elements in the promoter regions and the involvement of the identified MPKs in various cellular processes, as analyzed using Pathway Studio, suggest a role for the banana MPK gene family in diverse functions related to growth, development, and the stress response. This report is the first concerning the identification of members of a gene family and the elucidation of their role in various processes using the Musa Genome database. PMID:24275941

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

    PubMed

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

    2014-09-01

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

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

    PubMed Central

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

    2011-01-01

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

  12. The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain.

    PubMed

    Ma, Kai-Ge; Lv, Jia; Hu, Xiao-Dan; Shi, Li-Li; Chang, Ke-Wei; Chen, Xin-Lin; Qian, Yi-Hua; Yang, Wei-Na; Qu, Qiu-Min

    2016-07-01

    Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo. PMID:27163530

  13. Global Analysis of Serine/Threonine and Tyrosine Protein Phosphatase Catalytic Subunit Genes in Neurospora crassa Reveals Interplay Between Phosphatases and the p38 Mitogen-Activated Protein Kinase

    PubMed Central

    Ghosh, Arit; Servin, Jacqueline A.; Park, Gyungsoon; Borkovich, Katherine A.

    2013-01-01

    Protein phosphatases are integral components of the cellular signaling machinery in eukaryotes, regulating diverse aspects of growth and development. The genome of the filamentous fungus and model organism Neurospora crassa encodes catalytic subunits for 30 protein phosphatase genes. In this study, we have characterized 24 viable N. crassa phosphatase catalytic subunit knockout mutants for phenotypes during growth, asexual development, and sexual development. We found that 91% of the mutants had defects in at least one of these traits, whereas 29% possessed phenotypes in all three. Chemical sensitivity screens were conducted to reveal additional phenotypes for the mutants. This resulted in the identification of at least one chemical sensitivity phenotype for 17 phosphatase knockout mutants, including novel chemical sensitivities for two phosphatase mutants lacking a growth or developmental phenotype. Hence, chemical sensitivity or growth/developmental phenotype was observed for all 24 viable mutants. We investigated p38 mitogen-activated protein kinase (MAPK) phosphorylation profiles in the phosphatase mutants and identified nine potential candidates for regulators of the p38 MAPK. We demonstrated that the PP2C class phosphatase pph-8 (NCU04600) is an important regulator of female sexual development in N. crassa. In addition, we showed that the Δcsp-6 (ΔNCU08380) mutant exhibits a phenotype similar to the previously identified conidial separation mutants, Δcsp-1 and Δcsp-2, that lack transcription factors important for regulation of conidiation and the circadian clock. PMID:24347630

  14. A Mitogen-Activated Protein Kinase Pathway Essential for Mating and Contributing to Vegetative Growth in Neurospora crassa

    PubMed Central

    Li, Dan; Bobrowicz, Piotr; Wilkinson, Heather H.; Ebbole, Daniel J.

    2005-01-01

    MAP kinases homologous to Saccharomyces cerevisiae Fus3p/Kss1p have been identified in plant pathogenic fungi and are required for pathogenicity and sexual reproduction. To better understand the role of MAP kinase signaling in Neurospora crassa, and to identify downstream target genes of the pathway, we isolated, cloned, and disrupted the FUS3 homolog mak-2. Ste12p is a transcription factor target of Fus3p that activates genes of the mating pathway in yeast, and we also characterized the N. crassa STE12 homolog pp-1. The mak-2 and pp-1 mutants have reduced growth rate, produce short aerial hyphae, and fail to develop protoperithecia. In addition, ascospores carrying null mutations of either gene are inviable. Subtractive cloning was used to isolate genes having reduced expression in the mak-2 mutant. Expression of some of these genes is protoperithecia specific and three of them are part of a gene cluster potentially involved in the production of a polyketide secondary metabolite. Microarray analysis was used to extend the analysis of gene expression in mak-2 and pp-1 mutants. The role of the MAP kinase pathway in both sexual and asexual development as well as secondary metabolism is consistent with the dual regulation of the mating process and pathogencity observed in fungal pathogens.

  15. Immunolocalization of the mitogen-activated protein kinases p42MAPK and JNK1, and their regulatory kinases MEK1 and MEK4, in adult rat central nervous system.

    PubMed

    Flood, D G; Finn, J P; Walton, K M; Dionne, C A; Contreras, P C; Miller, M S; Bhat, R V

    1998-08-31

    Cell survival, death, and stress signals are transduced from the cell surface to the cytoplasm and nucleus via a cascade of phosphorylation events involving the mitogen-activated protein kinase (MAPK) family. We compared the distribution of p42 mitogen-activated protein kinase (p42MAPK) and its activator MAPK or ERK kinase (MEK1; involved in transduction of growth and differentiation signals), with c-Jun N-terminal kinase (JNK1) and its activator MEK4 (involved in transduction of stress and death signals) in the adult rat central nervous system. All four kinases were present in the cytoplasm, dendrites, and axons of neurons. The presence of p42MAPK and JNK1 in dendrites and axons, as well as in cell bodies, suggests a role for these kinases in phosphorylation and regulation of cytoplasmic targets. A high degree of correspondence was found between the regional distribution of MEK1 and p42MAPK. Immunostaining for MEK1 and p42MAPK was intense in olfactory structures, neocortex, hippocampus, striatum, midline, and interlaminar thalamic nuclei, hypothalamus, brainstem, Purkinje cells, and spinal cord. In addition to neurons, p42MAPK was also present in oligodendrocytes. Whereas MEK4 was ubiquitously distributed, JNK1 was more selective. Immunostaining for MEK4 and JNK1 was intense in the olfactory bulb, lower cortical layers, the cholinergic basal forebrain, most nuclei of the thalamus, medial habenula, and cranial motor nuclei. The distribution of MEK1 and p42MAPK proteins only partially overlapped with that of MEK4 and JNK1. This suggests that the growth/differentiation and death/stress pathways affected by these kinases may not necessarily act to counterbalance each other in response to extracellular stimuli. The differential distribution of these kinases may control the specificity of neuronal function to extracellular signals. PMID:9714150

  16. A Mitogen-Activated Protein Kinase Cascade Module, MKK3-MPK6 and MYC2, Is Involved in Blue Light-Mediated Seedling Development in Arabidopsis[C][W

    PubMed Central

    Sethi, Vishmita; Raghuram, Badmi; Sinha, Alok Krishna; Chattopadhyay, Sudip

    2014-01-01

    Mitogen-activated protein kinase (MAPK) pathways are involved in several signal transduction processes in eukaryotes. Light signal transduction pathways have been extensively studied in plants; however, the connection between MAPK and light signaling pathways is currently unknown. Here, we show that MKK3-MPK6 is activated by blue light in a MYC2-dependent manner. MPK6 physically interacts with and phosphorylates a basic helix-loop-helix transcription factor, MYC2, and is phosphorylated by a MAPK kinase, MKK3. Furthermore, MYC2 binds to the MPK6 promoter and regulates its expression in a feedback regulatory mechanism in blue light signaling. We present mutational and physiological studies that illustrate the function of the MKK3-MPK6-MYC2 module in Arabidopsis thaliana seedling development and provide a revised mechanistic view of photomorphogenesis. PMID:25139007

  17. Short communication: Camel milk ameliorates inflammatory responses and oxidative stress and downregulates mitogen-activated protein kinase signaling pathways in lipopolysaccharide-induced acute respiratory distress syndrome in rats.

    PubMed

    Zhu, Wei-Wei; Kong, Gui-Qing; Ma, Ming-Ming; Li, Yan; Huang, Xiao; Wang, Li-Peng; Peng, Zhen-Yi; Zhang, Xiao-Hua; Liu, Xiang-Yong; Wang, Xiao-Zhi

    2016-01-01

    Acute respiratory distress syndrome (ARDS) is a complex syndrome disorder with high mortality rate. Camel milk (CM) contains antiinflammatory and antioxidant properties and protects against numerous diseases. This study aimed to demonstrate the function of CM in lipopolysaccharide (LPS)-induced ARDS in rats. Camel milk reduced the lung wet:dry weight ratio and significantly reduced LPS-induced increases in neutrophil infiltration, interstitial and intra-alveolar edema, thickness of the alveolar wall, and lung injury scores of lung tissues. It also had antiinflammatory and antioxidant effects on LPS-induced ARDS. After LPS stimulation, the levels of proinflammatory cytokines (tumor necrosis factor-α, IL-10, and IL-1β) in serum and oxidative stress markers (malondialdehyde, myeloperoxidase, and total antioxidant capacity) in lung tissue were notably attenuated by CM. Camel milk also downregulated mitogen-activated protein kinase signaling pathways. Given these results, CM is a potential complementary food for ARDS treatment. PMID:26601576

  18. Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

    PubMed Central

    McLees, A; Graham, A; Malarkey, K; Gould, G W; Plevin, R

    1995-01-01

    In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7741705

  19. Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

    PubMed

    McLees, A; Graham, A; Malarkey, K; Gould, G W; Plevin, R

    1995-05-01

    In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line. PMID:7741705

  20. Cysteinyl-leukotrienes are released from astrocytes and increase astrocyte proliferation and glial fibrillary acidic protein via cys-LT1 receptors and mitogen-activated protein kinase pathway.

    PubMed

    Ciccarelli, Renata; D'Alimonte, Iolanda; Santavenere, Clara; D'Auro, Mariagrazia; Ballerini, Patrizia; Nargi, Eleonora; Buccella, Silvana; Nicosia, Simonetta; Folco, Giancarlo; Caciagli, Francesco; Di Iorio, Patrizia

    2004-09-01

    Cysteinyl-leukotrienes (cys-LTs), potent mediators in inflammatory diseases, are produced by nervous tissue, but their cellular source and role in the brain are not very well known. In this report we have demonstrated that rat cultured astrocytes express the enzymes (5'-lipoxygenase and LTC(4) synthase) required for cys-LT production, and release cys-LTs in resting condition and, to a greater extent, in response to calcium ionophore A23187, 1 h combined oxygen-glucose deprivation or 2-methyl-thioATP, a selective P2Y(1)/ATP receptor agonist. MK-886, a LT synthesis inhibitor, prevented basal and evoked cys-LT release. In addition, 2-methyl-thioATP-induced cys-LT release was abolished by suramin, a P2 receptor antagonist, or by inhibitors of ATP binding cassette proteins involved in cys-LT release. We also showed that astrocytes express cys-LT(1) and not cys-LT(2) receptors. The stimulation of these receptors by LTD(4) activated the mitogen-activated protein kinase (MAPK) pathway. This effect was: (i) insensitive to inhibitors of receptor-coupled Gi protein (pertussis toxin) or tyrosine kinase receptors (genistein); (ii) abolished by MK-571, a cys-LT(1) selective receptor antagonist, or PD98059, a MAPK inhibitor; (iii) reduced by inhibitors of calcium/calmodulin-dependent kinase II (KN-93), Ca(2+)-dependent and -independent (GF102903X) or Ca(2+)-dependent (Gö6976) protein kinase C isoforms. LTD(4) also increased astrocyte proliferation and glial fibrillary acidic protein content, which are considered hallmarks of reactive astrogliosis. Both effects were counteracted by cell pretreatment with MK-571 or PD98059. Thus, cys-LTs released from astrocytes might play an autocrine role in the induction of reactive astrogliosis that, in brain injuries, contributes to the formation of a reparative glial scar. PMID:15355318

  1. Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes

    PubMed Central

    Ramey, Guillemette; Deschemin, Jean-Christophe; Vaulont, Sophie

    2009-01-01

    Background The circulating hormone hepcidin plays a central role in iron homeostasis. Our goal was to establish an ex vivo iron-sensing model and to characterize the molecular mechanisms linking iron to hepcidin. Design and Methods Murine hepatocytes were isolated by the collagenase method, either from wild type or HFE knockout mice, and cultured 42 h without serum before treatments. Results After 42 h of serum-free culture, hepcidin gene expression was undetectable in the hepatocytes. Hepcidin gene expression could, however, be re-activated by an additional 24 h of incubation with 10% serum. Interestingly, addition of 30 μM holotransferrin consistently increased serum-dependent hepcidin levels 3- to 5-fold. The effects of serum and serum+holotransferrin were direct, transcriptional, independent of de novo protein synthesis and required the presence of bone morphogenetic protein. Transferrin receptor-2 activation by its ligand holotransferrin led to extracellular signal regulated kinase (ERK)/mitogen activated protein kinase pathway stimulation and the ERK specific inhibitor U0-126 blunted holotransferrin-mediated induction of hepcidin. ERK activation by holotransferrin provoked increased levels of phospho-Smad1/5/8 highlighting cross-talk between the bone morphogenetic protein/hemojuvelin and ERK1/2 pathways. Finally, we demonstrated, using hepatocytes isolated from Hfe−/− mice, that HFE was not critical for the hepcidin response to holotransferrin but important for basal hepcidin expression. Conclusions We demonstrate that hepatocytes are liver iron-sensor cells and that transferrin receptor-2, by signaling through the ERK1/2 pathway, and bone morphogenetic protein/hemojuvelin, by signaling through the Smad pathways, coordinately regulate the iron-sensing machinery linking holotransferrin to hepcidin. PMID:19454495

  2. The Kinase Activity-deficient Isoform of the Protein Araf Antagonizes Ras/Mitogen-activated Protein Kinase (Ras/MAPK) Signaling in the Zebrafish Embryo*

    PubMed Central

    Xiong, Cong; Liu, Xingfeng; Meng, Anming

    2015-01-01

    Raf kinases are important components of the Ras-Raf-Mek-Erk pathway and also cross-talk with other signaling pathways. Araf kinase has been demonstrated to inhibit TGF-β/Smad2 signaling by directly phosphorylating and accelerating degradation of activated Smad2. In this study, we show that the araf gene expresses in zebrafish embryos to produce a shorter transcript variant, araf-tv2, in addition to the full-length variant araf-tv1. araf-tv2 is predicted to encode a C-terminally truncated peptide without the kinase activity domain. Araf-tv2 can physically associate with Araf-tv1 but does not antagonize the inhibitory effect of Araf-tv1 on TGF-β/Smad2 signaling. Instead, Araf-tv2 interacts strongly with Kras and Nras, ultimately blocking MAPK activation by these Ras proteins. In zebrafish embryos, overexpression of araf-tv2 is sufficient to inhibit Fgf/Ras-promoted Erk activation, mesodermal induction, dorsal development, and neuroectodermal posteriorization. Therefore, different isoforms of Araf may participate in similar developmental processes but by regulating different signaling pathways. PMID:26306042

  3. Induction of Viral, 7-Methyl-Guanosine Cap-Independent Translation and Oncolysis by Mitogen-Activated Protein Kinase-Interacting Kinase-Mediated Effects on the Serine/Arginine-Rich Protein Kinase

    PubMed Central

    Brown, Michael C.; Bryant, Jeffrey D.; Dobrikova, Elena Y.; Shveygert, Mayya; Bradrick, Shelton S.; Chandramohan, Vidyalakshmi; Bigner, Darell D.

    2014-01-01

    ABSTRACT Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m7G) “cap”-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. IMPORTANCE We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. PMID:25187541

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

    PubMed

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

    2013-04-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Delayed Cell Cycle Progression in Selenoprotein W-depleted Cells Is Regulated by a Mitogen-activated Protein Kinase Kinase 4-p38/c-Jun NH2-terminal Kinase-p53 Pathway*

    PubMed Central

    Hawkes, Wayne Chris; Alkan, Zeynep

    2012-01-01

    Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a transient p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser-33 in p53, which is associated with decreased p53 ubiquitination and stabilization of p53. We report here that delayed cell cycle progression, Ser-33 phosphorylation, and p53 nuclear accumulation from SEPW1 depletion require mitogen-activated protein kinase kinase 4 (MKK4). Silencing MKK4 rescued G1 arrest, Ser-33 phosphorylation, and nuclear accumulation of p53 induced by SEPW1 depletion, but silencing MKK3, MKK6, or MKK7 did not. SEPW1 silencing did not change the phosphorylation state of MKK4 but increased total MKK4 protein. Silencing p38γ, p38δ, or JNK2 partially rescued G1 arrest from SEPW1 silencing, suggesting they signal downstream from MKK4. These results imply that SEPW1 silencing increases MKK4, which activates p38γ, p38δ, and JNK2 to phosphorylate p53 on Ser-33 and cause a transient G1 arrest. PMID:22730327

  7. p38β Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261.

    PubMed

    Beenstock, Jonah; Melamed, Dganit; Mooshayef, Navit; Mordechay, Dafna; Garfinkel, Benjamin P; Ahn, Natalie G; Admon, Arie; Engelberg, David

    2016-05-15

    Many enzymes are self-regulated and can either inhibit or enhance their own catalytic activity. Enzymes that do both are extremely rare. Many protein kinases autoactivate by autophosphorylating specific sites at their activation loop and are inactivated by phosphatases. Although mitogen-activated protein kinases (MAPKs) are usually activated by dual phosphorylation catalyzed by MAPK kinases (MAPKKs), the MAPK p38β is exceptional and is capable of self-activation by cis autophosphorylation of its activation loop residue T180. We discovered that p38β also autophosphorylates in trans two previously unknown sites residing within a MAPK-specific structural element known as the MAPK insert: T241 and S261. Whereas phosphorylation of T180 evokes catalytic activity, phosphorylation of S261 reduces the activity of T180-phosphorylated p38β, and phosphorylation of T241 reduces its autophosphorylation in trans Both phosphorylations do not affect the activity of dually phosphorylated p38β. T241 of p38β is found phosphorylated in vivo in bone and muscle tissues. In myogenic cell lines, phosphorylation of p38β residue T241 is correlated with differentiation to myotubes. T241 and S261 are also autophosphorylated in intrinsically active variants of p38α, but in this protein, they probably play a different role. We conclude that p38β is an unusual enzyme that automodulates its basal, MAPKK-independent activity by several autophosphorylation events, which enhance and suppress its catalytic activity. PMID:26976637

  8. Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

    PubMed Central

    Choi, T; Rulong, S; Resau, J; Fukasawa, K; Matten, W; Kuriyama, R; Mansour, S; Ahn, N; Vande Woude, G F

    1996-01-01

    Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8643471

  9. Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7.

    PubMed Central

    Fleming, Y; Armstrong, C G; Morrice, N; Paterson, A; Goedert, M; Cohen, P

    2000-01-01

    Stress-activated protein kinase 1 (SAPK1), also called c-Jun N-terminal kinase (JNK), becomes activated in vivo in response to pro-inflammatory cytokines or cellular stresses. Its full activation requires the phosphorylation of a threonine and a tyrosine residue in a Thr-Pro-Tyr motif, which can be catalysed by the protein kinases mitogen-activated protein kinase kinase (MKK)4 and MKK7. Here we report that MKK4 shows a striking preference for the tyrosine residue (Tyr-185), and MKK7 a striking preference for the threonine residue (Thr-183) in three SAPK1/JNK1 isoforms tested (JNK1 alpha 1, JNK2 alpha 2 and JNK3 alpha 1). For this reason, MKK4 and MKK7 together produce a synergistic increase in the activity of each SAPK1/JNK isoform in vitro. The MKK7 beta variant, which is several hundred-fold more efficient in activating all three SAPK1/JNK isoforms than is MKK7 alpha', is equally specific for Thr-183. MKK7 also phosphorylates JNK2 alpha 2 at Thr-404 and Ser-407 in vitro, Ser-407 being phosphorylated much more rapidly than Thr-183 in vitro. Thr-404/Ser-407 are phosphorylated in unstimulated human KB cells and HEK-293 cells, and phosphorylation is increased in response to an osmotic stress (0.5 M sorbitol). However, in contrast with Thr-183 and Tyr-185, the phosphorylation of Thr-404 and Ser-407 is not increased in response to other agonists that activate MKK7 and SAPK1/JNK, suggesting that phosphorylation of these residues is catalysed by another protein kinase, such as CK2, which also phosphorylates Thr-404 and Ser-407 in vitro. MKK3, MKK4 and MKK6 all show a strong preference for phosphorylation of the tyrosine residue of the Thr-Gly-Tyr motifs in their known substrates SAPK2a/p38, SAPK3/p38 gamma and SAPK4/p38 delta. MKK7 also phosphorylates SAPK2a/p38 at a low rate (but not SAPK3/p38 gamma or SAPK4/p38 delta), and phosphorylation occurs exclusively at the tyrosine residue, demonstrating that MKK7 is intrinsically a 'dual-specific' protein kinase. PMID:11062067

  10. Hemoglobin Receptor Protein from Porphyromonas gingivalis Induces Interleukin-8 Production in Human Gingival Epithelial Cells through Stimulation of the Mitogen-Activated Protein Kinase and NF-κB Signal Transduction Pathways

    PubMed Central

    Fujita, Yuki; Nakayama, Masaaki; Naito, Mariko; Yamachika, Eiki; Inoue, Tetsuyoshi; Nakayama, Koji; Iida, Seiji

    2014-01-01

    Periodontitis is an inflammatory disease of polymicrobial origin affecting the tissues supporting the tooth. The oral anaerobic bacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, triggers a series of host inflammatory responses that promote the destruction of periodontal tissues. Among the virulence factors of P. gingivalis, hemoglobin receptor protein (HbR) is a major protein found in culture supernatants. In this study, we investigated the roles of HbR in the production of inflammatory mediators. We found that HbR induced interleukin-8 (IL-8) production in the human gingival epithelial cell line Ca9-22. p38 mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (Erk1/2) were activated in HbR-stimulated Ca9-22 cells. Inhibitors of p38 MAPK (SB203580) and Erk1/2 (PD98059) blocked HbR-induced IL-8 production. Additionally, HbR stimulated the translocation of NF-κB-p65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-κB pathway. In addition, small interfering RNA (siRNA) targeting activating transcription factor 2 (ATF-2) or cyclic AMP-response element-binding protein (CREB) inhibited HbR-induced IL-8 production. Moreover, pretreatment with SB203580 and PD98059 reduced HbR-induced phosphorylation of CREB and ATF-2, respectively. Combined pretreatment with an inhibitor of NF-κB (BAY11-7082) and SB203580 was more efficient in inhibiting the ability of HbR to induce IL-8 production than pretreatment with either BAY11-7082 or SB203580 alone. Thus, in Ca9-22 cells, the direct activation of p38 MAPK and Erk1/2 by HbR caused the activation of the transcription factors ATF-2, CREB, and NF-κB, thus resulting in the induction of IL-8 production. PMID:24126532

  11. The investigation of Mitogen-Activated Protein kinase Phosphatase-1 as a potential pharmacological target in non-small cell lung carcinomas, assisted by non-invasive molecular imaging

    PubMed Central

    2010-01-01

    Background Invasiveness and metastasis are the most common characteristics of non small cell lung cancer (NSCLC) and causes of tumour-related morbidity and mortality. Mitogen-activated protein kinases (MAPKs) signalling pathways have been shown to play critical roles in tumorigenesis. However, the precise pathological role(s) of mitogen-activated protein kinase phosphatase-1 (MKP-1) in different cancers has been controversial such that the up-regulation of MKP-1 in different cancers does not always correlate to a better prognosis. In this study, we showed that the induction of MKP-1 lead to a significant retardation of proliferation and metastasis in NSCLC cells. We also established that rosiglitazone (a PPARγ agonist) elevated MKP-1 expression level in NSCLC cells and inhibited tumour metastasis. Methods Both wildtype and dominant negative forms of MKP-1 were constitutively expressed in NSCLC cell line H441GL. The migration and invasion abilities of these cells were examined in vitro. MKP-1 modulating agents such as rosiglitazone and triptolide were used to demonstrate MKP-1's role in tumorigenesis. Bioluminescent imaging was utilized to study tumorigenesis of MKP-1 over-expressing H441GL cells and anti-metastatic effect of rosiglitazone. Results Over-expression of MKP-1 reduced NSCLC cell proliferation rate as well as cell invasive and migratory abilities, evident by the reduced expression levels of MMP-2 and CXCR4. Mice inoculated with MKP-1 over-expressing H441 cells did not develop NSCLC while their control wildtype H441 inoculated littermates developed NSCLC and bone metastasis. Pharmacologically, rosiglitazone, a peroxisome proliferator activated receptor-γ (PPARγ) agonist appeared to induce MKP-1 expression while reduce MMP-2 and CXCR4 expression. H441GL-inoculated mice receiving daily oral rosiglitazone treatment demonstrated a significant inhibition of bone metastasis when compared to mice receiving sham treatment. We found that rosiglitazone treatment

  12. Quantitative measurement of epidermal growth factor receptor-mitogen-activated protein kinase signal transduction using a nine-plex, peptide-based immunoassay.

    PubMed

    Rauh-Adelmann, Christine; Moskow, John M; Graham, James R; Yen, Lucy G; Boucher, Jeffrey I; Murphy, Cheryl E; Nadler, Timothy K; Gordon, Neal F; Radding, Jeffrey A

    2008-04-15

    Aberrant epidermal growth factor receptor (EGFR, ErbB1) signaling is implicated in cell transformation, motility, and invasion in a variety of cell types, and EGFR is the target of several anticancer drugs. However, the kinetics of EGFR signaling and the individual contributions of site-specific phosphorylation events remain largely unknown. A peptide-based, multiplex immunoassay approach was developed to simultaneously measure both total and phosphorylated protein in a single sample. The approach involves the proteolytic digestion of proteins prior to the isolation and quantitation of site-specific phosphorylation events within an individual protein. Quantitation of phosphorylated and total proteins, in picomolar to nanomolar concentrations, were interpolated from standard curves generated with synthetic peptides that correspond to the peptide targets used in the immunoassays. In this study, a bead-based, nine-plex immunoassay measuring total and phosphorylated protein was constructed to measure temporal, site-specific phosphorylation of key members of the EGFR pathway (ErbB1 receptor, MEK1, MEK2, ERK1, and ERK2) in A431 cells stimulated with epidermal growth factor. The effect of MEK inhibition on this pathway was determined using a known MEK kinase inhibitor, SL327. The results reported herein are the first quantitative measurements of site-specific phosphorylation events and total proteins in a single sample, at the same time representing a new paradigm for standardized protein and phosphorylation analysis using multiplexed, peptide-based, sandwich immunoassays. PMID:18275835

  13. Mitogen-activated protein kinase signal transduction and DNA repair network are involved in aluminum-induced DNA damage and adaptive response in root cells of Allium cepa L.

    PubMed Central

    Panda, Brahma B.; Achary, V. Mohan M.

    2014-01-01

    In the current study, we studied the role of signal transduction in aluminum (Al3+)-induced DNA damage and adaptive response in root cells of Allium cepa L. The root cells in planta were treated with Al3+ (800 μM) for 3 h without or with 2 h pre-treatment of inhibitors of mitogen-activated protein kinase (MAPK), and protein phosphatase. Also, root cells in planta were conditioned with Al3+ (10 μM) for 2 h and then subjected to genotoxic challenge of ethyl methane sulfonate (EMS; 5 mM) for 3 h without or with the pre-treatment of the aforementioned inhibitors as well as the inhibitors of translation, transcription, DNA replication and repair. At the end of treatments, roots cells were assayed for cell death and/or DNA damage. The results revealed that Al3+ (800 μM)-induced significant DNA damage and cell death. On the other hand, conditioning with low dose of Al3+ induced adaptive response conferring protection of root cells from genotoxic stress caused by EMS-challenge. Pre-treatment of roots cells with the chosen inhibitors prior to Al3+-conditioning prevented or reduced the adaptive response to EMS genotoxicity. The results of this study suggested the involvement of MAPK and DNA repair network underlying Al-induced DNA damage and adaptive response to genotoxic stress in root cells of A. cepa. PMID:24926302

  14. Regulation of cyclooxygenase-2 and cytosolic phospholipase A2 gene expression by lipopolysaccharide through the RNA-binding protein HuR: involvement of NADPH oxidase, reactive oxygen species and mitogen-activated protein kinases

    PubMed Central

    Lin, Wei-Ning; Lin, Chih-Chung; Cheng, Hsin-Yi; Yang, Chuen-Mao

    2011-01-01

    BACKGROUND AND PURPOSE Lipopolysaccharide (LPS)-induced expression of cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) has been implicated in several respiratory diseases. HuR is known to enhance the expression of genes by binding to 3′-untranslated region (3′-UTR) of mRNA and stabilizing mRNA. However, the exact mechanisms by which HuR affects the stability of mRNA and modulates LPS-induced COX-2 and cPLA2 expression in human tracheal smooth muscle cells (HTSMCs) are not known. EXPERIMENTAL APPROACH The expression of prostaglandin E2 (PGE2) was measured by ELISA, and pro-inflammatory proteins were determined by use of a promoter assay, PCR or Western blot analysis. Overexpression of siRNAs to knock down the target components was used to manipulate the expression of HuR. Release of reactive oxygen species (ROS) was detected by fluorescence dye. The activation of signalling components was assessed by comparing phosphorylation levels, localization of protein kinases or coimmunoprecipitation assay. KEY RESULTS LPS induced COX-2 and cPLA2 expression via post-translational regulation of mRNA stabilization, which were attenuated by transfection with HuR siRNA in HTSMCs. In addition, LPS-stimulated NADPH oxidase activation and ROS generation were attenuated by the NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and apocynin (APO). Generation of ROS induced phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK and JNK1/2, which was attenuated by DPI and APO and the ROS scavenger N-acetylcysteine. CONCLUSIONS AND IMPLICATIONS These results suggested that in HTSMCs, LPS-induced COX-2 and cPLA2 expression is mediated through NADPH oxidase/ROS-dependent MAPKs associated with HuR accumulation in the cytoplasm. Activated MAPKs may regulate the nucleocytoplasmic shuttling of HuR, and thus induce the cytoplasmic accumulation of HuR. PMID:21391979

  15. Exposure to Cerium Oxide Nanoparticles Is Associated With Activation of Mitogen-activated Protein Kinases Signaling and Apoptosis in Rat Lungs

    PubMed Central

    Rice, Kevin M.; Nalabotu, Siva K.; Manne, Nandini D.P.K.; Kolli, Madhukar B.; Nandyala, Geeta; Arvapalli, Ravikumar; Ma, Jane Y.; Blough, Eric R.

    2015-01-01

    Objectives: With recent advances in nanoparticle manufacturing and applications, potential exposure to nanoparticles in various settings is becoming increasing likely. No investigation has yet been performed to assess whether respiratory tract exposure to cerium oxide (CeO2) nanoparticles is associated with alterations in protein signaling, inflammation, and apoptosis in rat lungs. Methods: Specific-pathogen-free male Sprague-Dawley rats were instilled with either vehicle (saline) or CeO2 nanoparticles at a dosage of 7.0 mg/kg and euthanized 1, 3, 14, 28, 56, or 90 days after exposure. Lung tissues were collected and evaluated for the expression of proteins associated with inflammation and cellular apoptosis. Results: No change in lung weight was detected over the course of the study; however, cerium accumulation in the lungs, gross histological changes, an increased Bax to Bcl-2 ratio, elevated cleaved caspase-3 protein levels, increased phosphorylation of p38 MAPK, and diminished phosphorylation of ERK-1/2-MAPK were detected after CeO2 instillation (p<0.05). Conclusions: Taken together, these data suggest that high-dose respiratory exposure to CeO2 nanoparticles is associated with lung inflammation, the activation of signaling protein kinases, and cellular apoptosis, which may be indicative of a long-term localized inflammatory response. PMID:26081650

  16. Low-frequency stimulation induces a durable long-term depression in young adult hyperthyroid rats: the role of p38 mitogen-activated protein kinase and protein phosphatase 1.

    PubMed

    Tan, Burak; Bitiktaş, Soner; Kavraal, Şehrazat; Dursun, Nurcan; Dönmez Altuntaş, Hamiyet; Suer, Cem

    2016-06-15

    Long-term potentiation and long-term depression (LTD) are cellular mechanisms of learning and memory in the mammalian brain. We have previously shown that adult hyperthyroid rats showed a delay in the acquisition of a place learning task and attenuated long-term potentiation. However, changes in LTD in hyperthyroidism remain unclear. Rats were administered 0.2 mg/kg/day of L-thyroxine for 21 days starting at postnatal day 40 to induce hyperthyroidism. LTD was induced in the dentate gyrus using low-frequency stimulation (LFS) of the perforant pathway. The mRNA expressions of p38 mitogen-activated protein kinase (p38-MAPK) and protein phosphatase 1 (PP1) were evaluated using a quantitative reverse transcriptase PCR. In control rats, a standard LFS protocol induced a slight depression of the population spike (PS) amplitude during the induction phase of LTD (76±13% vs. baseline), but a small potentiation of the PS amplitude was observed in the early (107±18%) and late (111±20%) phases of LTD. Interestingly, in the hyperthyroid rats, the same LFS protocol induced a reliable LTD in the dentate gyrus of the hippocampus as evidenced by a marked depression in the PS amplitude during the induction (54±6% vs. baseline) and the early phases (56±8%) of LTD. Elevated mRNA levels of p38-MAPK and PP1 were observed in the hippocampus of the LFS-treated hyperthyroid rats compared with the hippocampus of the vehicle-treated hyperthyroid rats. No significant change in p38-MAPK or PP1 mRNA expression was observed in the euthyroid rats. The present study shows that a standard LFS protocol can induce a durable depression of synaptic strength and an upregulation of PP1 and p38-MAPK mRNA in hyperthyroid rats. We conclude that hyperthyroidism can induce molecular changes associated with degeneration of the hippocampus. The relationship between the levels of thyroid hormone and dementia requires further investigation. PMID:27128724

  17. Toll-Like Receptor- and Filarial Antigen-Mediated, Mitogen-Activated Protein Kinase- and NF-κB-Dependent Regulation of Angiogenic Growth Factors in Filarial Lymphatic Pathology

    PubMed Central

    Anuradha, R.; Kumar, N. Pavan; George, P. Jovvian; Kumaraswami, V.; Nutman, Thomas B.

    2012-01-01

    Filarial lymphatic pathology is of multifactorial origin, with inflammation, lymphangiogenesis, and innate immune responses all playing important roles. The role of Toll-like receptors (TLRs) in the development of filarial pathology is well characterized. Similarly, the association of pathology with elevated levels of plasma angiogenic factors has also been documented. To examine the association between TLR function and the development of lymphangiogenesis in filarial infections, we examined TLR- and filarial antigen-induced expression and production of various angiogenic growth factors. We demonstrate that TLR ligands (specifically TLR2, -3, and -5 ligands) induce significantly increased expression/production of vascular endothelial growth factor A (VEGF-A) and angiopoietin-1 (Ang-1) in the peripheral blood mononuclear cells of individuals with lymphatic pathology (CP individuals) compared to that in cells of asymptomatic infected (INF) individuals. Similarly, filarial antigens induce significantly enhanced production of VEGF-C in CP compared with INF individuals. TLR2-mediated enhancement of angiogenic growth factor production in CP individuals was shown to be dependent on mitogen-activated protein kinase (MAPK) and NF-κB signaling, as pharmacologic inhibition of either extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, or NF-κB signaling resulted in significantly diminished production of VEGF-A and Ang-1. Our data therefore strongly suggest an important association between TLR signaling and lymphangiogenesis in the development of pathology in human lymphatic filariasis. PMID:22508858

  18. Design, Synthesis, and Anti-inflammatory Properties of Orally Active 4-(Phenylamino)-pyrrolo[2,1-f][1,2,4]triazine p38[alpha] Mitogen-Activated Protein Kinase Inhibitors

    SciTech Connect

    Hynes, Jr., John; Dyckman, Alaric J.; Lin, Shuqun; Wrobleski, Stephen T.; Wu, Hong; Gillooly, Kathleen M.; Kanner, Steven B.; Lonial, Herinder; Loo, Derek; McIntyre, Kim W.; Pitt, Sidney; Shen, Ding Ren; Shuster, David J.; Yang, XiaoXia; Zhang, Rosemary; Behnia, Kamelia; Zhang, Hongjian; Marathe, Punit H.; Doweyko, Arthur M.; Tokarski, John S.; Sack, John S.; Pokross, Matthew; Kiefer, Susan E.; Newitt, John A.; Barrish, Joel C.; Dodd, John; Schieven, Gary L.; Leftheris, Katerina

    2008-06-30

    A novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors consisting of substituted 4-(phenylamino)-pyrrolo[2,1- f][1,2,4]triazines has been discovered. An initial subdeck screen revealed that the oxindole-pyrrolo[2,1- f][1,2,4]triazine lead 2a displayed potent enzyme inhibition (IC 50 60 nM) and was active in a cell-based TNFalpha biosynthesis inhibition assay (IC 50 210 nM). Replacement of the C4 oxindole with 2-methyl-5- N-methoxybenzamide aniline 9 gave a compound with superior p38 kinase inhibition (IC 50 10 nM) and moderately improved functional inhibition in THP-1 cells. Further replacement of the C6 ester of the pyrrolo[2,1- f][1,2,4]triazine with amides afforded compounds with increased potency, excellent oral bioavailability, and robust efficacy in a murine model of acute inflammation (murine LPS-TNFalpha). In rodent disease models of chronic inflammation, multiple compounds demonstrated significant inhibition of disease progression leading to the advancement of 2 compounds 11b and 11j into further preclinical and toxicological studies.

  19. Enhanced IL-1{beta}-induced IL-8 production in cystic fibrosis lung epithelial cells is dependent of both mitogen-activated protein kinases and NF-{kappa}B signaling

    SciTech Connect

    Muselet-Charlier, Celine; Roque, Telma; Boncoeur, Emilie; Chadelat, Katarina; Clement, Annick; Jacquot, Jacky; Tabary, Olivier . E-mail: olivier.tabary@st-antoine.inserm.fr

    2007-06-01

    Transcription nuclear factor-{kappa}B (NF-{kappa}B) is hyperactivated in cystic fibrosis (CF) lung epithelial cells, and participates in exaggerated IL-8 production in the CF lung. We recently found that rapid activation of NF-{kappa}B occurred in a CF lung epithelial IB3-1 cell line (CF cells) upon IL-1{beta} stimulation, which was not observed in its CFTR-corrected lung epithelial S9 cell line (corrected cells). To test whether other signaling pathways such as that of mitogen-activated protein kinases (MAPKs) could be involved in IL-1{beta}-induced IL-8 production of CF cells, we investigated ERK1/2, JNK, and p38MAP signaling compared to NF-{kappa}B. Within 30 min, exposure to IL-1{beta} caused high activation of NF-{kappa}B, ERK1/2, p38MAP but not JNK in CF cells compared to corrected cells. Treatment of IL-1{beta}-stimulated CF cells with a series of chemical inhibitors of NF-{kappa}B, ERK1/2, and p38MAP, when used separately, reduced slightly IL-8 production. However, when used together, these inhibitors caused a blockade in IL-1{beta}-induced IL-8 production in CF cells. Understanding of the cross-talk between NF-{kappa}B and MAPKs signaling in CF lung epithelial cells may help in developing new therapeutics to reduce lung inflammation in patients with CF.

  20. Antinociceptive effects of analgesic-antitumor peptide (AGAP), a neurotoxin from the scorpion Buthus martensii Karsch, on formalin-induced inflammatory pain through a mitogen-activated protein kinases-dependent mechanism in mice.

    PubMed

    Mao, Qinghong; Ruan, Jiaping; Cai, Xueting; Lu, Wuguang; Ye, Juan; Yang, Jie; Yang, Yang; Sun, Xiaoyan; Cao, Junli; Cao, Peng

    2013-01-01

    In the present study, we investigated the anti-nociceptive effect and the underlying mechanism of the analgesic-antitumor peptide (AGAP), a neurotoxin from the scorpion Buthus martensii Karsch. AGAP in doses of 0.2, 1 and 5 µg was injected intraplantarly (i.pl.) before formalin injection 10 min at the same site. The suppression by intraplantar injection of AGAP on formalin-induced spontaneous nociceptive behaviors was investigated. The results show that AGAP could dose-dependently inhibit formalin-induced two-phase spontaneous flinching response. To investigate the mechanism of action of treatment with AGAP in inflammatory pain, the expressions of peripheral and spinal phosphorylated mitogen-activated protein kinases (phospho-MAPKs) including p-p38, p-ERK and p-JNK were examined. We found that formalin increased the expressions of peripheral and spinal MAPKs, which were prevented by pre-intraplantar injection of AGAP in inflammation pain model in mice. AGAP could also decrease the expression of spinal Fos induced by formalin. Furthermore, combinations the lower doses of the inhibitors of MAPKs (U0126, SP600125, or SB203580 0.1 µg) with the lower dose of AGAP (0.2 µg), the results suggested that AGAP could potentiate the effects of the inhibitors of MAPKs on the inflammatory pain. The present results indicate that pre-intraplantar injection of AGAP prevents the inflammatory pain induced by formalin through a MAPKs-mediated mechanism in mice. PMID:24244296

  1. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

  2. Luteolin prevents solar radiation-induced matrix metalloproteinase-1 activation in human fibroblasts: a role for p38 mitogen-activated protein kinase and interleukin-20 released from keratinocytes.

    PubMed

    Wölfle, Ute; Heinemann, Anja; Esser, Philipp R; Haarhaus, Birgit; Martin, Stefan F; Schempp, Christoph M

    2012-10-01

    Human skin is continuously exposed to solar radiation, which can result in photoaging, a process involving both dermal and, to a lesser extent, epidermal structures. Previously, we have shown that the flavonoid luteolin protects the epidermis from ultraviolet (UV)-induced damage by a combination of UV-absorbing, antioxidant, and antiinflammatory properties. The aim of the present study was to determine direct and indirect effects of luteolin on dermal fibroblasts as major targets of photoaging. Stimulation of fibroblasts with UVA light or the proinflammatory cytokine interleukin-20 (IL-20) is associated with wrinkled skin, increased IL-6 secretion, matrix metalloproteinase (MMP-1) expression, and hyaluronidase activity. All of these targets were inhibited by luteolin via interference with the p38 mitogen-activated protein kinase (MAPK) pathway. Next, we assessed the role of conditioned supernatants from keratinocytes irradiated with solar-simulated radiation (SSR) on nonirradiated dermal fibroblasts. In keratinocytes, luteolin inhibited SSR-induced production of IL-20, also via interference with the p38 MAPK pathway. Similarly, keratinocyte supernatant-induced IL-6 and MMP-1 expression in fibroblasts was reduced by pretreatment of keratinocytes with luteolin. Finally, these results were confirmed ex vivo on skin explants treated with luteolin before UV irradiation. Our results suggest that SSR-mediated production of soluble factors in keratinocytes is modulated by luteolin and may attenuate photoaging in dermal fibroblasts. PMID:23004935

  3. Luteolin Prevents Solar Radiation-Induced Matrix Metalloproteinase-1 Activation in Human Fibroblasts: A Role for p38 Mitogen-Activated Protein Kinase and Interleukin-20 Released from Keratinocytes

    PubMed Central

    Heinemann, Anja; Esser, Philipp R.; Haarhaus, Birgit; Martin, Stefan F.; Schempp, Christoph M.

    2012-01-01

    Abstract Human skin is continuously exposed to solar radiation, which can result in photoaging, a process involving both dermal and, to a lesser extent, epidermal structures. Previously, we have shown that the flavonoid luteolin protects the epidermis from ultraviolet (UV)-induced damage by a combination of UV-absorbing, antioxidant, and antiinflammatory properties. The aim of the present study was to determine direct and indirect effects of luteolin on dermal fibroblasts as major targets of photoaging. Stimulation of fibroblasts with UVA light or the proinflammatory cytokine interleukin-20 (IL-20) is associated with wrinkled skin, increased IL-6 secretion, matrix metalloproteinase (MMP-1) expression, and hyaluronidase activity. All of these targets were inhibited by luteolin via interference with the p38 mitogen-activated protein kinase (MAPK) pathway. Next, we assessed the role of conditioned supernatants from keratinocytes irradiated with solar-simulated radiation (SSR) on nonirradiated dermal fibroblasts. In keratinocytes, luteolin inhibited SSR-induced production of IL-20, also via interference with the p38 MAPK pathway. Similarly, keratinocyte supernatant-induced IL-6 and MMP-1 expression in fibroblasts was reduced by pretreatment of keratinocytes with luteolin. Finally, these results were confirmed ex vivo on skin explants treated with luteolin before UV irradiation. Our results suggest that SSR-mediated production of soluble factors in keratinocytes is modulated by luteolin and may attenuate photoaging in dermal fibroblasts. PMID:23004935

  4. Inhibition of mitogen-activated protein kinases/nuclear factor κB-dependent inflammation by a novel chalcone protects the kidney from high fat diet-induced injuries in mice.

    PubMed

    Fang, Qilu; Deng, Liancheng; Wang, Lintao; Zhang, Yali; Weng, Qiaoyou; Yin, Haimin; Pan, Yong; Tong, Chao; Wang, Jingying; Liang, Guang

    2015-11-01

    The prevalence of obesity has increased dramatically worldwide leading to increases in obesity-related complications, such as obesity-related glomerulopathy (ORG). Obesity is a state of chronic, low-grade inflammation, and increased inflammation in the adipose and kidney tissues has been shown to promote the progression of renal damage in obesity. Current therapeutic options for ORG are fairly limited and, as a result, we are seeing increased rates of progression to end-stage renal disease. Chalcones are a class of naturally occurring compounds with various pharmacological properties. 1-(3,4-Dihydroxyphenyl)-3-(2-methoxyphenyl)prop-2-en-1-one (L2H17) is a chalcone that we have previously synthesized and found capable of inhibiting the lipopolysaccharide-induced inflammatory response in macrophages. In this study, we investigated L2H17's effect on obesity-induced renal injury using palmitic acid-induced mouse peritoneal macrophages and high fat diet-fed mice. Our results indicate that L2H17 protects against renal injury through the inhibition of the mitogen-activated protein kinase/nuclear factor κB pathways significantly by decreasing the expression of proinflammatory cytokines and cell adhesion molecules and improving kidney histology and pathology. These findings lead us to believe that L2H17, as an anti-inflammatory agent, can be a potential therapeutic option in treating ORG. PMID:26354992

  5. Glycyrrhizic acid prevents ultraviolet-B-induced photodamage: a role for mitogen-activated protein kinases, nuclear factor kappa B and mitochondrial apoptotic pathway.

    PubMed

    Afnan, Quadri; Kaiser, Peerzada J; Rafiq, Rather A; Nazir, Lone A; Bhushan, Shashi; Bhardwaj, Subhash C; Sandhir, Rajat; Tasduq, Sheikh A

    2016-06-01

    Glycyrrhizic acid (GA), a natural triterpene, has received attention as an agent that has protective effects against chronic diseases including ultraviolet UV-B-induced skin photodamage. However, the mechanism of its protective effect remains elusive. Here, we used an immortalized human keratinocyte cell line (HaCaT) and a small animal model (BALB/c mice), to investigate the protective effects of GA against UV-B-induced oxidative damage, and additionally, delineated the molecular mechanisms involved in the UV-B-mediated inflammatory and apoptotic response. In the HaCaT cells, GA inhibited the UV-B-mediated increase in intracellular reactive oxygen species (ROS) and down-regulated the release of pro-inflammatory cytokines interleukin (IL)-1α, -1β and -6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2). GA inhibited UV-B-mediated activation of p38 and JNK MAP kinases, COX-2 expression and nuclear translocation of NF-κB. Furthermore, GA inhibited UV-B-mediated apoptosis by attenuating translocation of Bax from the cytosol to mitochondria, thus preserving mitochondrial integrity. GA-treated HaCaT cells also exhibited elevated antiapoptotic Bcl-2 protein, concomitant with reduced caspase-3 cleavage and decreased PARP-1 protein. In BALB/c mice, topical application of GA on dorsal skin exposed to UV-B irradiation protected against epidermal hyperplasia, lymphocyte infiltration and expression of several inflammatory proteins, p38, JNK, COX-2, NF-κB and ICAM-1. Based on the above findings, we conclude that GA protects against UV-B-mediated photodamage by inhibiting the signalling cascades triggered by oxidative stress, including MAPK/NF-κB activation, as well as apoptosis. Thus, GA has strong potential to be used as a therapeutic/cosmeceutical agent against photodamage. PMID:26836460

  6. Angiotensin II regulates ACE and ACE2 in neurons through p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 signaling

    PubMed Central

    Xiao, Liang; Haack, Karla K. V.

    2013-01-01

    Brain ANG II plays an important role in modulating sympathetic function and homeostasis. The generation and degradation of ANG II are carried out, to a large extent, through the angiotensin-converting enzyme (ACE) and ACE2, respectively. In disease states, such as hypertension and chronic heart failure, central expression of ACE is upregulated and ACE2 is decreased in central sympathoregulatory neurons. In this study, we determined the expression of ACE and ACE2 in response to ANG II in a neuronal cell culture and the subsequent signaling mechanism(s) involved. A mouse catecholaminergic neuronal cell line (CATH.a) was treated with ANG II (30, 100, and 300 nM) for 24 h, and protein expression was determined by Western blot analysis. ANG II induced a significant dose-dependent increase in ACE and decrease in ACE2 mRNA and protein expression in CATH.a neurons. This effect was abolished by pretreatment of the cells with the p38 MAPK inhibitor SB-203580 (10 μM) 30 min before administration of ANG II or the ERK1/2 inhibitor U-0126 (10 μM). These data suggest that ANG II increases ACE and attenuates ACE2 expression in neurons via the ANG II type 1 receptor, p38 MAPK, and ERK1/2 signaling pathways. PMID:23535237

  7. Involvement of mitogen-activated protein kinase and NF-κB signaling pathways in perfluorooctane sulfonic acid-induced inflammatory reaction in BV2 microglial cells.

    PubMed

    Zhu, Jingying; Qian, Wenyi; Wang, Yixin; Gao, Rong; Wang, Jun; Xiao, Hang

    2015-12-01

    Microglial activation is closely related to the pathogenesis of neurodegenerative diseases by producing proinflammatory cytokines. Perfluorooctane sulfonic acid (PFOS), known as an emerging persistent organic pollutant, is reported to disturb human immune homeostasis; however, whether it affects cytokine production or the immune response in the central nervous system remains unclear. The present study was aimed to explore whether PFOS contributed to inflammatory action and to investigate the corresponding mechanisms in BV2 microglia. PFOS-mediated morphologic changes, cytokine responses and signaling events were examined by light microscopy, real-time polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot assays. Our results indicated that PFOS increased BV2 cells activation and simultaneously increased tumor necrosis factor alpha and interleukin-6 expression. In addition, the c-Jun N-terminal protein kinase inhibitor (SP600125), as well as ERK1/2 blocker (PD98059), transcriptionally at least, displayed anti-inflammatory properties on PFOS-elicited cytokine responses. Moreover, the inflammatory transcription factor NF-κB was specifically activated by PFOS as well. These results, taken together, suggested that PFOS exerts its functional effects on the response of microglial cell activation via, in part, the c-Jun N-terminal protein kinase, ERK and NF-κB signaling pathways with its subsequent influence on proinflammatory action. PMID:25677194

  8. 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. PMID:26668315

  9. The effect of RGS12 on PDGFbeta receptor signalling to p42/p44 mitogen activated protein kinase in mammalian cells.

    PubMed

    Sambi, Balwinder S; Hains, Melinda D; Waters, Catherine M; Connell, Michelle C; Willard, Francis S; Kimple, Adam J; Pyne, Susan; Siderovski, David P; Pyne, Nigel J

    2006-07-01

    We have previously shown that the PDGFbeta receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of p42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFbeta receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFbeta receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of p42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFbeta receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFbeta receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFbeta receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFbeta receptor in ASM cells. PMID:16214305

  10. Allicin induces apoptosis of the MGC-803 human gastric carcinoma cell line through the p38 mitogen-activated protein kinase/caspase-3 signaling pathway.

    PubMed

    Zhang, Xuecheng; Zhu, Yong; Duan, Wei; Feng, Chen; He, Xuan

    2015-04-01

    Gastric cancer is one of the most common forms of malignant tumor, and the development of anti‑gastric cancer drugs with minimal toxicity is of clinical importance. Allicin is extracted from Allium sativum (garlic). Recent research, including clinical experiments, has shown that garlic has anticancer and tumor suppressive effects. The present study aimed to investigate the effects of allicin on the MGC‑803 human gastric carcinoma cell line, and to further explore the possible mechanisms of its tumor suppressor effects. The effects of allicin on the MGC‑803 cells were initially examined using an 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. Hoechst staining was also used, in order to demonstrate the impact of allicin on MGC‑803 cell apoptosis. In addition, western blot analysis was performed to determine the abnormal expression levels of apoptosis‑associated proteins, following the treatment of MGC‑803 cells with allicin. Western blotting was also used to investigate the specific mechanisms underlying allicin‑induced apoptosis of MGC‑803 cells. The rate of MGC‑803 apoptosis was significantly increased, when the concentration and treatment time of allicin were increased. Hoechst staining detected an enhanced rate of apoptosis, and enhanced expression levels of cleaved caspase 3 were determined by western blotting. Notably, the protein expression levels of p38 were increased when the MGC‑803 cells were treated with allicin. The results of the present study suggest that allicin may inhibit the proliferation and induce the apoptosis of MGC‑803 human gastric carcinoma cells, and this may partially be achieved through the enhanced expression of p38 and cleaved caspase 3. PMID:25523417

  11. Characterization of Mitogen-Activated Protein Kinase Expression in Nucleus Accumbens and Hippocampus of Rats Subjected to Food Selection in the Cafeteria Diet Protocol.

    PubMed

    Sarro-Ramírez, Andrea; Sánchez, Daniel; Tejeda-Padrón, Alma; Buenfil-Canto, Linda Vianey; Valladares-García, Jorge; Pacheco-Pantoja, Elda; Arias-Carrión, Oscar; Murillo-Rodríguez, Eric

    2016-01-01

    Obesity is a world-wide health problem that requires different experimental perspectives to understand the onset of this disease, including the neurobiological basis of food selection. From a molecular perspective, obesity has been related with activity of several endogenous molecules, including the mitogenactivated protein kinases (MAP-K). The aim of this study was to characterize MAP-K expression in hedonic and learning and memory brain-associated areas such as nucleus accumbens (AcbC) and hippocampus (HIPP) after food selection. We show that animals fed with cafeteria diet during 14 days displayed an increase in p38 MAP-K activity in AcbC if chose cheese. Conversely, a diminution was observed in animals that preferred chocolate in AcbC. Also, a decrease of p38 MAP-K phosphorylation was found in HIPP in rats that selected either cheese or chocolate. Our data demonstrate a putative role of MAP-K expression in food selection. These findings advance our understanding of neuromolecular basis engaged in obesity. PMID:27071784

  12. p38 mitogen-activated protein kinase inhibition modulates nucleus pulposus cell apoptosis in spontaneous resorption of herniated intervertebral discs: An experimental study in rats.

    PubMed

    Zhu, Yu; Liu, Jin-Tao; Yang, Li-Yan; Du, Wen-Pei; Li, Xiao-Chun; Qian, Xiang; Yu, Peng-Fei; Liu, Jian-Wen; Jiang, Hong

    2016-05-01

    The present study was performed to investigate the role of p38 mitogen‑activated protein kinase (MAPK) in the resorption of herniated intervertebral discs in 30 rats. In the non‑contained and p38 MAPK inhibition (p38i) groups, two coccygeal intervertebral discs (IVDs) were removed and wounded prior to relocation into the subcutaneous space of the skin of the back. In the contained group, the cartilage endplates maintained their integrity. Furthermore, SB203580 was injected intraperitoneally into the p38i group, whereas saline was injected into the other two groups. In the non‑contained group, the weight of the relocated IVDs decreased to a greater extent over time when compared with the contained and p38i groups. Phosphorylated p38, tumor necrosis factor‑α, and interleukin‑1β were observed to exhibit higher expression levels in the non‑contained group compared with the contained and p38i groups, at weeks 1 and 4 post‑surgery. The expression level of caspase‑3 and the densities of apoptotic disc cells were significantly higher in the non‑contained group compared with the contained and p38i groups at 4 weeks post‑surgery. In conclusion, p38 MAPK induces apoptosis in IVDs, while also accelerating the resorption of the relocated IVDs. Thus, p38 MAPK may be important in spontaneous resorption of IVDs. PMID:27035219

  13. Exercise preconditioning reduces neonatal incision surgery-induced enhanced hyperalgesia via inhibition of P38 mitogen-activated protein kinase and IL-1β, TNF-α release.

    PubMed

    Gong, Xingrui; Jiang, Jing; Zhang, Mazhong

    2016-08-01

    Neonatal surgery leads to enhanced hyperalgesia to noxious stimulation in adulthood via a mechanism caused by enhanced phosphorylated (p)-p38 expression in microglia. We tested the effect of exercise on reducing enhanced hypersensitivity primed by neonatal incision surgery. Adult female Wistar rats, with or without neonatal incision surgery at postnatal day (P) 3, received right hind paw plantar incision surgery under anesthesia at P44. The rats performed wheel-running exercise from P22 to P41. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured and ipsilateral spinal cords were collected for protein quantification. For PWT and PWL, exercise reduced the pain index after incision surgery at P44 in rats with neonatal surgery (P<0.01). Western blots showed that exercise suppressed P-p38 expression relative to adult rats without neonatal surgery (P<0.05). Results of ELISA showed that exercise reduced IL-1β and TNF-α (P<0.05) concentration in the ipsilateral spinal cord. Exercise preconditioning is an effective approach to reducing enhanced adult hyperalgesia primed by neonatal surgery. The mechanism may be explained by exercise-induced inhibition of P-p38 activation and IL-1β, TNF-α release. PMID:27235543

  14. P38 mitogen-activated protein kinase activity is required during mitosis for timely satisfaction of the mitotic checkpoint but not for the fidelity of chromosome segregation.

    PubMed

    Lee, Kyunghee; Kenny, Alison E; Rieder, Conly L

    2010-07-01

    Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by approximately 40% in nontransformed human RPE-1, approximately 80% in PtK2 (rat kangaroo), and approximately 25% in mouse cells, and this prolongation leads to an elevated mitotic index. However, under this condition chromatid segregation and cytokinesis are normal. Using Mad2/YFP-expressing cells, we show the prolongation of mitosis in the absence of p38 activity is directly due to a delay in satisfying the mitotic checkpoint. Inhibiting p38 did not affect the rate of chromosome motion; however, it did lead to the formation of significantly (10%) longer metaphase spindles. From these data we conclude that normal p38 activity is required for the timely stable attachment of all kinetochores to spindle microtubules, but not for the fidelity of the mitotic process. We speculate that p38 activity promotes timely checkpoint satisfaction by indirectly influencing those motor proteins (e.g., Klp10, Klp67A) involved in regulating the dynamics of kinetochore microtubule ends. PMID:20462950

  15. Activation of the Classical Mitogen-Activated Protein Kinases Is Part of the Shiga Toxin-Induced Ribotoxic Stress Response and May Contribute to Shiga Toxin-Induced Inflammation

    PubMed Central

    Ahluwalia, Amrita; Schimmel, Jennifer J.; Rogers, Arlin B.; Leong, John M.; Thorpe, Cheleste M.

    2015-01-01

    Infection with enterohemorrhagic Escherichia coli (EHEC) can result in severe disease, including hemorrhagic colitis and the hemolytic uremic syndrome. Shiga toxins (Stx) are the key EHEC virulence determinant contributing to severe disease. Despite inhibiting protein synthesis, Shiga toxins paradoxically induce the expression of proinflammatory cytokines from various cell types in vitro, including intestinal epithelial cells (IECs). This effect is mediated in large part by the ribotoxic stress response (RSR). The Shiga toxin-induced RSR is known to involve the activation of the stress-activated protein kinases (SAPKs) p38 and JNK. In some cell types, Stx also can induce the classical mitogen-activated protein kinases (MAPKs) or ERK1/2, but the mechanism(s) by which this activation occurs is unknown. In this study, we investigated the mechanism by which Stx activates ERK1/2s in IECs and the contribution of ERK1/2 activation to interleukin-8 (IL-8) expression. We demonstrate that Stx1 activates ERK1/2 in a biphasic manner: the first phase occurs in response to StxB1 subunit, while the second phase requires StxA1 subunit activity. We show that the A subunit-dependent ERK1/2 activation is mediated through ZAK-dependent signaling, and inhibition of ERK1/2 activation via the MEK1/2 inhibitors U0126 and PD98059 results in decreased Stx1-mediated IL-8 mRNA. Finally, we demonstrate that ERK1/2 are activated in vivo in the colon of Stx2-intoxicated infant rabbits, a model in which Stx2 induces a primarily neutrophilic inflammatory response. Together, our data support a role for ERK1/2 activation in the development of Stx-mediated intestinal inflammation. PMID:26483408

  16. Alteration of energy metabolism gene expression in cumulus cells affects oocyte maturation via MOS-mitogen-activated protein kinase pathway in dairy cows with an unfavorable "Fertil-" haplotype of one female fertility quantitative trait locus.

    PubMed

    Brisard, Daphné; Desmarchais, Alice; Touzé, Jean-Luc; Lardic, Lionel; Freret, Sandrine; Elis, Sebastien; Nuttinck, Fabienne; Camous, Sylvaine; Dupont, Joelle; Uzbekova, Svetlana

    2014-03-01

    Prim'Holstein heifers selected for the "Fertil-" homozygous haplotype of QTL-Female-Fert ility-BTA3 showed a greater rate of early pregnancy failure and slower embryo development after IVM suggesting lower oocyte quality than those selected for "Fertile+". We aimed to ascertain intrafollicular factors related to lower oocyte quality in "Fertil-" cows. Analysis of individual oocytes showed meiotic progression delay in "Fertil-" compared with "Fertil+" dairy cows after in vivo maturation and IVM (P < 0.05). Expression of several genes localized to QTL-F-Fert-BTA3 or related to meiosis and mitogen-activated protein kinase pathway was analyzed in individual metaphase-II oocytes using reverse transcription- real-time polymerase chain reaction. Energy metabolism, apoptosis, extracellular matrix, and QTL-F-Fert-BTA3 genes were analyzed in surrounding cumulus cells (CC). In vivo, a significant decrease in prostaglandin synthase PTGES1 and PTGS2 expression coupled with lower PTGS2 protein abundance in CC and reduced expression of MOS in enclosed metaphase-II oocytes from "Fertil-" cows was observed. IVM strongly deregulated gene expression in CC and in oocytes compared with in vivo; nevertheless, differential expression of several genes including PEX19, NAMPT and MOS was observed between the two haplotypes. During IVM, PTGS2 activity inhibitor NS398 (50 μM) led to lower expression of fatty acid synthase (FASN) in CC and of MOS in treated metaphase-II oocytes. Using immunofluorescence, MOS protein was localized to a midbody-like contractile ring separating the polar body from the ooplasm, suggesting a role in the terminal stage of oocyte maturation. Our results suggest that factors involved in prostaglandin synthesis and lipid metabolism in CC could impair oocyte maturation, and might be involved in the reduced fertility of "Fertil-" cows. PMID:24377862

  17. Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases.

    PubMed Central

    Sudbeck, B D; Baumann, P; Ryan, G J; Breitkopf, K; Nischt, R; Krieg, T; Mauch, C

    1999-01-01

    Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation. PMID:10085241

  18. Suppression of Mitochondrial Biogenesis through Toll-Like Receptor 4–Dependent Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling in Endotoxin-Induced Acute Kidney Injury

    PubMed Central

    Smith, Joshua A.; Stallons, L. Jay; Collier, Justin B.; Chavin, Kenneth D.

    2015-01-01

    Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiologic feature of sepsis-induced acute kidney injury (AKI), the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)-induced AKI. Downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of MB, was noted at the mRNA level at 3 hours and protein level at 18 hours in the renal cortex, and was associated with loss of renal function after LPS treatment. LPS-mediated suppression of PGC-1α led to reduced expression of downstream regulators of MB and electron transport chain proteins along with a reduction in renal cortical mitochondrial DNA content. Mechanistically, Toll-like receptor 4 (TLR4) knockout mice were protected from renal injury and disruption of MB after LPS exposure. Immunoblot analysis revealed activation of tumor progression locus 2/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (TPL-2/MEK/ERK) signaling in the renal cortex by LPS. Pharmacologic inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was associated with a reduction in proinflammatory cytokine (e.g., tumor necrosis factor-α [TNF-α], interleukin-1β) expression at 3 hours after LPS exposure. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal dysfunction, after LPS-induced AKI. Finally, systemic administration of recombinant tumor necrosis factor-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis. PMID:25503387

  19. Andrographolide stimulates p38 mitogen-activated protein kinase-nuclear factor erythroid-2-related factor 2-heme oxygenase 1 signaling in primary cerebral endothelial cells for definite protection against ischemic stroke in rats.

    PubMed

    Yen, Ting-Lin; Chen, Ray-Jade; Jayakumar, Thanasekaran; Lu, Wan-Jung; Hsieh, Cheng-Ying; Hsu, Ming-Jen; Yang, Chih-Hao; Chang, Chao-Chien; Lin, Yen-Kuang; Lin, Kuan-Hung; Sheu, Joen-Rong

    2016-04-01

    Stroke pathogenesis involves complex oxidative stress-related pathways. The nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) pathways have been considered molecular targets in pharmacologic intervention for ischemic diseases. Andrographolide, a labdane diterpene, has received increasing attention in recent years because of its various pharmacologic activities. We determined that andrographolide modulates the mitogen-activated protein kinase (MAPK)-Nrf2-HO-1 signaling cascade in primary cerebral endothelial cells (CECs) to provide positive protection against middle cerebral artery occlusion (MCAO)-induced ischemic stroke in rats. In the present study, andrographolide (10 μM) increased HO-1 protein and messenger RNA expressions, Nrf2 phosphorylation, and nuclear translocation in CECs, and these activities were disrupted by a p38 MAPK inhibitor, SB203580, but not by the extracellular signal-regulated kinase inhibitor PD98059 or c-Jun amino-terminal kinase inhibitor SP600125. Similar results were observed in confocal microscopy analysis. Moreover, andrographolide-induced Nrf2 and HO-1 protein expressions were significantly inhibited by Nrf2 small interfering RNA. Moreover, HO-1 knockdown attenuated the protective effect of andrographolide against oxygen-glucose deprivation-induced CEC death. Andrographolide (0.1 mg/kg) significantly suppressed free radical formation, blood-brain barrier disruption, and brain infarction in MCAO-insulted rats, and these effects were reversed by the HO-1 inhibitor zinc protoporphyrin IX. The mechanism is attributable to HO-1 activation, as directly evidenced by andrographolide-induced pronounced HO-1 expression in brain tissues, which was highly localized in the cerebral capillary. In conclusion, andrographolide increased Nrf2-HO-1 expression through p38 MAPK regulation, confirming that it provides protection against MCAO-induced brain injury. These findings provide strong evidence that andrographolide could

  20. Metformin-mediated downregulation of p38 mitogen-activated protein kinase-dependent excision repair cross-complementing 1 decreases DNA repair capacity and sensitizes human lung cancer cells to paclitaxel.

    PubMed

    Tseng, Sheng-Chieh; Huang, Yu-Ching; Chen, Huang-Jen; Chiu, Hsien-Chun; Huang, Yi-Jhen; Wo, Ting-Yu; Weng, Shao-Hsing; Lin, Yun-Wei

    2013-02-15

    Metformin, an extensively used and well-tolerated drug for treating individuals with type 2 diabetes, has recently gained significant attention as an anticancer drug. On the other hand, paclitaxel (Taxol) is a new antineoplastic drug that has shown promise in the treatment of non-small cell lung cancer (NSCLC). High expression levels of excision repair cross-complementary 1 (ERCC1) in cancers have been positively associated with the DNA repair capacity and a poor prognosis in NSCLC patients treated with platinum-containing chemotherapy. In this current study, paclitaxel was found to increase phosphorylation of mitogen-activated protein kinase (MAPK) kinase 3/6 (MKK3/6)-p38 MAPK as well as protein and mRNA levels of ERCC1 in H1650 and H1703 cells. Moreover, paclitaxel-induced ERCC1 protein and mRNA levels significantly decreased via the downregulation of p38 activity by either a p38 MAPK inhibitor SB202190 or p38 knockdown with specific small interfering RNA (siRNA). Specific inhibition of ERCC1 with siRNA was found to enhance the paclitaxel-induced cytotoxic effect and growth inhibition. Furthermore, metformin was able to not only decrease the paclitaxel-induced p38 MAPK-mediated ERCC1 expression, but also augment the cytotoxic effect induced by paclitaxel. Finally, expression of constitutive activate MKK6 or HA-p38 MAPK vectors in lung cancer cells was able to abrogate ERCC1 downregulation by metformin and paclitaxel as well as cell viability and DNA repair capacity. Overall, our results suggest that inhibition of the p38 MAPK signaling by metformin coupled with paclitaxel therapy in human NSCLC cells may be a clinically useful combination, which however will require further validation. PMID:23228696

  1. Sann-Joong-Kuey-Jian-Tang induces autophagy in HepG2 cells via regulation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and p38 mitogen-activated protein kinase pathways

    PubMed Central

    CHUANG, WAN-LING; SU, CHIN-CHENG; LIN, PING-YI; LIN, CHI-CHEN; CHEN, YAO-LI

    2015-01-01

    Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer. PMID:25847489

  2. Role of the Cyclic AMP Response Element Binding Complex and Activation of Mitogen-Activated Protein Kinases in Synergistic Activation of the Glycoprotein Hormone α Subunit Gene by Epidermal Growth Factor and Forskolin

    PubMed Central

    Roberson, Mark S.; Ban, Makiko; Zhang, Tong; Mulvaney, Jennifer M.

    2000-01-01

    The aim of these studies was to elucidate a role for epidermal growth factor (EGF) signaling in the transcriptional regulation of the glycoprotein hormone α subunit gene, a subunit of chorionic gonadotropin. Studies examined the effects of EGF and the adenylate cyclase activator forskolin on the expression of a transfected α subunit reporter gene in a human choriocarcinoma cell line (JEG3). At maximal doses, administration of EGF resulted in a 50% increase in a subunit reporter activity; forskolin administration induced a fivefold activation; the combined actions of EGF and forskolin resulted in synergistic activation (greater than eightfold) of the α subunit reporter. Mutagenesis studies revealed that the cyclic AMP response elements (CRE) were required and sufficient to mediate EGF-forskolin-induced synergistic activation. The combined actions of EGF and forskolin resulted in potentiated activation of extracellular signal-regulated kinase (ERK) enzyme activity compared with EGF alone. Specific blockade of ERK activation was sufficient to block EGF-forskolin-induced synergistic activation of the α subunit reporter. Pretreatment of JEG3 cells with a p38 mitogen-activated protein kinase inhibitor did not influence activation of the α reporter. However, overexpression of c-Jun N-terminal kinase (JNK)-interacting protein 1 as a dominant interfering molecule abolished the synergistic effects of EGF and forskolin on the α subunit reporter. CRE binding studies suggested that the CRE complex consisted of CRE binding protein and EGF-ERK-dependent recruitment of c-Jun–c-Fos (AP-1) to the CRE. A dominant negative form of c-Fos (A-Fos) that specifically disrupts c-Jun–c-Fos DNA binding inhibited synergistic activation of the α subunit. Thus, synergistic activation of the α subunit gene induced by EGF-forskolin requires the ERK and JNK cascades and the recruitment of AP-1 to the CRE binding complex. PMID:10779323

  3. A Novel p38 Mitogen-activated Protein Kinase/Elk-1 Transcription Factor-dependent Molecular Mechanism Underlying Abnormal Endothelial Cell Proliferation in Plexogenic Pulmonary Arterial Hypertension*

    PubMed Central

    Patel, Monal; Predescu, Dan; Tandon, Rajive; Bardita, Cristina; Pogoriler, Jennifer; Bhorade, Sangeeta; Wang, Minhua; Comhair, Suzy; Ryan-Hemnes, Anna; Chen, Jiwang; Machado, Roberto; Husain, Aliya; Erzurum, Serpil; Predescu, Sanda

    2013-01-01

    Plexiform lesions (PLs), the hallmark of plexogenic pulmonary arterial hypertension (PAH), contain phenotypically altered, proliferative endothelial cells (ECs). The molecular mechanism that contributes to EC proliferation and formation of PLs is poorly understood. We now show that a decrease in intersectin-1s (ITSN-1s) expression due to granzyme B (GrB) cleavage during inflammation associated with PAH and the high p38/Erk1/2MAPK activity ratio caused by the GrB/ITSN cleavage products lead to EC proliferation and selection of a proliferative/plexiform EC phenotype. We used human pulmonary artery ECs of PAH subjects (ECPAH), paraffin-embedded and frozen human lung tissue, and animal models of PAH in conjunction with microscopy imaging, biochemical, and molecular biology approaches to demonstrate that GrB cleaves ITSN-1s, a prosurvival protein of lung ECs, and generates two biologically active fragments, an N-terminal fragment (GrB-EHITSN) with EC proliferative potential and a C-terminal product with dominant negative effects on Ras/Erk1/2. The proliferative potential of GrB-EHITSN is mediated via sustained phosphorylation of p38MAPK and Elk-1 transcription factor and abolished by chemical inhibition of p38MAPK. Moreover, lung tissue of PAH animal models and human specimens and ECPAH express lower levels of ITSN-1s compared with controls and the GrB-EHITSN cleavage product. Moreover, GrB immunoreactivity is associated with PLs in PAH lungs. The concurrent expression of the two cleavage products results in a high p38/Erk1/2MAPK activity ratio, which is critical for EC proliferation. Our findings identify a novel GrB-EHITSN-dependent pathogenic p38MAPK/Elk-1 signaling pathway involved in the poorly understood process of PL formation in severe PAH. PMID:23893408

  4. A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.

    PubMed

    Patel, Monal; Predescu, Dan; Tandon, Rajive; Bardita, Cristina; Pogoriler, Jennifer; Bhorade, Sangeeta; Wang, Minhua; Comhair, Suzy; Hemnes, Anna Ryan; Ryan-Hemnes, Anna; Chen, Jiwang; Machado, Roberto; Husain, Aliya; Erzurum, Serpil; Predescu, Sanda

    2013-09-01

    Plexiform lesions (PLs), the hallmark of plexogenic pulmonary arterial hypertension (PAH), contain phenotypically altered, proliferative endothelial cells (ECs). The molecular mechanism that contributes to EC proliferation and formation of PLs is poorly understood. We now show that a decrease in intersectin-1s (ITSN-1s) expression due to granzyme B (GrB) cleavage during inflammation associated with PAH and the high p38/Erk1/2(MAPK) activity ratio caused by the GrB/ITSN cleavage products lead to EC proliferation and selection of a proliferative/plexiform EC phenotype. We used human pulmonary artery ECs of PAH subjects (EC(PAH)), paraffin-embedded and frozen human lung tissue, and animal models of PAH in conjunction with microscopy imaging, biochemical, and molecular biology approaches to demonstrate that GrB cleaves ITSN-1s, a prosurvival protein of lung ECs, and generates two biologically active fragments, an N-terminal fragment (GrB-EH(ITSN)) with EC proliferative potential and a C-terminal product with dominant negative effects on Ras/Erk1/2. The proliferative potential of GrB-EH(ITSN) is mediated via sustained phosphorylation of p38(MAPK) and Elk-1 transcription factor and abolished by chemical inhibition of p38(MAPK). Moreover, lung tissue of PAH animal models and human specimens and EC(PAH) express lower levels of ITSN-1s compared with controls and the GrB-EH(ITSN) cleavage product. Moreover, GrB immunoreactivity is associated with PLs in PAH lungs. The concurrent expression of the two cleavage products results in a high p38/Erk1/2(MAPK) activity ratio, which is critical for EC proliferation. Our findings identify a novel GrB-EH(ITSN)-dependent pathogenic p38(MAPK)/Elk-1 signaling pathway involved in the poorly understood process of PL formation in severe PAH. PMID:23893408

  5. Isolation of Intrinsically Active (MEK-independent) Variants of the ERK Family of Mitogen-activated Protein (MAP) Kinases*S⃞♦

    PubMed Central

    Levin-Salomon, Vered; Kogan, Konstantin; Ahn, Natalie G.; Livnah, Oded; Engelberg, David

    2008-01-01

    MAPKs are key components of cell signaling pathways with a unique activation mechanism: i.e. dual phosphorylation of neighboring threonine and tyrosine residues. The ERK enzymes form a subfamily of MAPKs involved in proliferation, differentiation, development, learning, and memory. The exact role of each Erk molecule in these processes is not clear. An efficient strategy for addressing this question is to activate individually each molecule, for example, by expressing intrinsically active variants of them. However, such molecules were not produced so far. Here, we report on the isolation, via a specifically designed genetic screen, of six variants (each carries a point mutation) of the yeast MAPK Mpk1/Erk that are active, independent of upstream phosphorylation. One of the activating mutations, R68S, occurred in a residue conserved in the mammalian Erk1 (Arg-84) and Erk2 (Arg-65) and in the Drosophila ERK Rolled (Arg-80). Replacing this conserved Arg with Ser rendered these MAPKs intrinsically active to very high levels when tested in vitro as recombinant proteins. Combination of the Arg to Ser mutation with the sevenmaker mutation (producing Erk2R65S+D319N and RolledR80S+D334N) resulted in even higher activity (45 and 70%, respectively, in reference to fully active dually phosphorylated Erk2 or Rolled). Erk2R65S and Erk2R65S+D319N were found to be spontaneously active also when expressed in human HEK293 cells. We further revealed the mechanism of action of the mutants and show that it involves acquisition of autophosphorylation activity. Thus, a first generation of Erk molecules that are spontaneously active in vitro and in vivo has been obtained. PMID:18829462

  6. The Proteasome Inhibitor Bortezomib Enhances ATRA-Induced Differentiation of Neuroblastoma Cells via the JNK Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Luo, Peihua; Lin, Meili; Li, Lin; Yang, Bo; He, Qiaojun

    2011-01-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Differentiated human NBs are associated with better outcome and lower stage; induction of differentiation is considered to be therapeutically advantageous. All-trans retinoic acid (ATRA) has been shown to induce the differentiation of neuroblastoma (NB) cell lines. The proteasome inhibitor bortezomib inhibits cell growth and angiogenesis in NBs. Here, we investigated the synergistic effect between bortezomib and ATRA in inducing NB cell differentiation in different NB cell lines. Bortezomib combined with ATRA had a significantly enhanced antiproliferative effect. This inhibition was characterized by a synergistic increase in neuronal differentiation. At the same time, the combination therapy showed little neuronal toxicity which was assessed in primary cultures of rat cerebellar granule cells by the MTT assay, PI staining. The combination of bortezomib and ATRA triggered increased differentiation through the activation of proteins, including RARα, RARβ, RARγ, p-JNK and p21, compared with ATRA treatment alone. Using JNK inhibitor SP600125 to block JNK-dependent activity, the combination therapy-induced neuronal differentiation was partially attenuated. In addition, p21 shRNA had no effect on the combination therapy-induced neuronal differentiation. The in vivo antitumor activities were examined in human NB cell xenografts and GFP-labeled human NB cell xenografts. Treatment of human NB cell CHP126-bearing nude mice with ATRA plus bortezomib resulted in more significant tumor growth inhibition than mice treated with either drug alone. These findings provide the rationale for the development of a new therapeutic strategy for NB based on the pharmacological combination of ATRA and bortezomib. PMID:22087283

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2016-07-01

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

  9. Targeting Mitogen-Activated Protein Kinase/Extracellular Signal–Regulated Kinase Kinase in the Mutant (V600E) B-Raf Signaling Cascade Effectively Inhibits Melanoma Lung Metastases

    PubMed Central

    Sharma, Arati; Tran, Melissa A.; Liang, Shile; Sharma, Arun K.; Amin, Shantu; Smith, Charles D.; Dong, Cheng; Robertson, Gavin P.

    2009-01-01

    Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by V600EB-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)–mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal–regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant V600EB-Raf signaling cascade to inhibit melanoma metastases. PMID:16912199

  10. Mitogen-activated protein kinases regulate expression of neuronal nitric oxide synthase and neurite outgrowth via non-classical retinoic acid receptor signaling in human neuroblastoma SH-SY5Y cells.

    PubMed

    Fujibayashi, Tatsuya; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Shudo, Koichi; Katsuki, Hiroshi

    2015-10-01

    We have previously shown that retinoic acid receptor (RAR) stimulation by an agonist Am80 recruits nitric oxide-dependent signaling via increased expression of neuronal nitric oxide synthase (nNOS) in rat midbrain slice cultures. Using neuroblastoma SH-SY5Y cells, here we investigated the mechanisms of RAR-induced nNOS expression, together with relationship between nNOS expression and neurite outgrowth. Am80 promoted neurite outgrowth, which was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K; LY294002), c-Jun N-terminal kinase (JNK; SP600125) and p38 mitogen-activated protein kinase (p38 MAPK; SB203580). A selective nNOS inhibitor 3-bromo-nitroindazole also suppressed Am80-induced neurite outgrowth. Am80-induced increase in nNOS protein expression was attenuated by LY294002, SP600125 and SB203580, whereas increase in nNOS mRNA expression was attenuated only by LY294002. Am80-induced activation of JNK and p38 MAPK was blocked by LY294002, suggesting that these kinases acted downstream of PI3K. We also confirmed that DAX1, a nuclear receptor reported to regulate nNOS expression, was up-regulated in response to Am80. siRNA-mediated knockdown of DAX1 abrogated Am80-induced nNOS expression and neurite outgrowth. These results reveal for the first time that nNOS expression is crucial for RAR-mediated neurite outgrowth, and that non-genomic signaling such as JNK and p38 MAPK is involved in RAR-mediated nNOS expression. PMID:26422672

  11. Role of reactive oxygen species in brucein D-mediated p38-mitogen-activated protein kinase and nuclear factor-κB signalling pathways in human pancreatic adenocarcinoma cells

    PubMed Central

    Lau, S T; Lin, Z X; Leung, P S

    2010-01-01

    Background: In human pancreatic adenocarcinoma, nuclear factor-kappa-B (NF-κB) transcription factor is constitutively activated that contributes to the resistance of the tumour cells to induced apoptosis. In our earlier studies, we have shown that brucein D (BD) mediated apoptosis through activation of the p38-mitogen-activated protein kinase (MAPK) signalling pathway in pancreatic cancer cells. This study investigated the function of reactive oxygen species (ROS) in BD-mediated p38-MAPK and NF-κB signalling pathways in PANC-1 cells. Methods: Glutathione and dihydroethidium assays were used to measure the antioxidant and superoxide levels, respectively. The protein expression of p22phox, p67phox and p38-MAPK were examined by western blot. The NF-κB activity was evaluated by electrophoretic mobility shift assay. Results: Treatment with BD depleted the intracellular glutathione levels in PANC-1 cells. Brucein D triggered the activation of NADPH oxidase isoforms, p22phox and p67phox while enhancing the generation of superoxide. Increases in both intracellular ROS and NADPH oxidase activity were inhibited by an antioxidant, N-acetylcysteine (NAC). Brucein D-mediated activation of p38-MAPK was also inhibited by NAC. However, inhibition of NF-κB activity in BD-treated cells was independent of ROS. In vivo studies showed that BD treatment effectively reduced the rate of xenograft human pancreatic tumour in nude mice with no significant toxicity. Conclusion: These data suggest that BD is an apoptogenic agent for pancreatic cancer cells through activation of the redox-sensitive p38-MAPK pathway and inhibition of NF-κB anti-apoptotic activity in pancreatic cancer cells. PMID:20068565

  12. Anti-inflammatory Effects of Schisandra chinensis (Turcz.) Baill Fruit Through the Inactivation of Nuclear Factor-κB and Mitogen-activated Protein Kinases Signaling Pathways in Lipopolysaccharide-stimulated Murine Macrophages

    PubMed Central

    Kang, Young-Soon; Han, Min-Ho; Hong, Su-Hyun; Park, Cheol; Hwang, Hye-Jin; Kim, Byung Woo; Kyoung, Kim Ho; Choi, Young Whan; Kim, Cheol Min; Choi, Yung Hyun

    2014-01-01

    Background: Schisandrae Fructus, the dried fruit of Schisandra chinensis (Turcz.) Baill. (Magnoliaceae), is widely used in traditional medicine for the treatment of a number of chronic inflammatory diseases. This study examined the anti-inflammatory effects of Schisandrae Fructus ethanol extract (SF) on the production of pro-inflammatory substances in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods: To measure the effects of SF on pro-inflammatory mediator and inflammatory cytokine’s expression and production in RAW 264.7 cells, we used the following methods: cell viability assay, Griess reagent assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, Western blotting analysis and immunofluorescence staining. Results: Stimulation of the RAW 264.7 cells with LPS caused an elevated production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin (IL)-1β, which was markedly inhibited by the pretreatment with SF without causing any cytotoxic effects. SF also inhibited the expression of inducible NO synthase, TNF-α, and IL-1β protein and their mRNAs in LPS-stimulated RAW 264.7 cells. Furthermore, SF attenuated LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) by reducing inhibitory-κB degradation, and reduced the phosphorylation of mitogen-activated protein kinases (MAPKs), implying that SF regulated LPS-induced NF-κB-dependent inflammatory pathways through suppression of MAPKs activation. Conclusions: SF may be useful for the treatment of various inflammatory diseases. PMID:25574463

  13. Oxidative Stress Induces Premature Senescence by Stimulating Caveolin-1 Gene Transcription through p38 Mitogen-Activated Protein Kinase/Sp1–Mediated Activation of Two GC-Rich Promoter Elements

    PubMed Central

    Dasari, Arvind; Bartholomew, Janine N.; Volonte, Daniela; Galbiati, Ferruccio

    2015-01-01

    Cellular senescence is believed to represent a natural tumor suppressor mechanism. We have previously shown that up-regulation of caveolin-1 was required for oxidative stress–induced premature senescence in fibroblasts. However, the molecular mechanisms underlying caveolin-1 up-regulation in senescent cells remain unknown. Here, we show that subcytotoxic oxidative stress generated by hydrogen peroxide application promotes premature senescence and stimulates the activity of a (−1,296) caveolin-1 promoter reporter gene construct in fibroblasts. Functional deletion analysis mapped the oxidative stress response elements of the mouse caveolin-1 promoter to the sequences −244/−222 and −124/−101. The hydrogen peroxide–mediated activation of both Cav-1 (−244/−222) and Cav-1 (−124/−101) was prevented by the antioxidant quercetin. Combination of electrophoretic mobility shift studies, chromatin immunoprecipitation analysis, Sp1 overexpression experiments, as well as promoter mutagenesis identifies enhanced Sp1 binding to two GC-boxes at −238/−231 and −118/−106 as the core mechanism of oxidative stress–triggered caveolin-1 transactivation. In addition, signaling studies show p38 mitogen-activated protein kinase (MAPK) as the upstream regulator of Sp1-mediated activation of the caveolin-1 promoter following oxidative stress. Inhibition of p38 MAPK prevents the oxidant-induced Sp1-mediated up-regulation of caveolin-1 protein expression and development of premature senescence. Finally, we show that oxidative stress induces p38-mediated up-regulation of caveolin-1 and premature senescence in normal human mammary epithelial cells but not in MCF-7 breast cancer cells, which do not express caveolin-1 and undergo apoptosis. This study delineates for the first time the molecular mechanisms that modulate caveolin-1 gene transcription upon oxidative stress and brings new insights into the redox control of cellular senescence in both normal and cancer

  14. Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer's disease.

    PubMed

    Pei, Jin-Jing; Braak, Heiko; An, Wen-Lin; Winblad, Bengt; Cowburn, Richard F; Iqbal, Khalid; Grundke-Iqbal, Inge

    2002-12-30

    The abnormal hyperphosphorylation of tau in Alzheimer's disease (AD) has been proposed to involve the extracellular-signal-regulated protein kinase (ERK) of the mitogen-activated protein (MAP) kinase family. ERK is phosphorylated and thereby activated by MAP kinase kinase (MEK). In the present study, we determined the intracellular and regional distribution of the active forms of both MEK1/2 and ERK1/2, i.e. p-MEK1/2 and p-ERK1/2 in the entorhinal, hippocampal, and temporal cortices of 49 brains staged for neurofibrillary changes according to Braak and Braak's protocol. We found that p-MEK1/2 and p-ERK1/2 were present in the initial stages of neurofibrillary degeneration in the projecting neurons in the transentorhinal region, and extended into other brain regions co-incident with the progressive sequence of neurofibrillary changes up to and including Braak stage VI. It appeared that the accumulation of p-MEK1/2 and p-ERK1/2 was initiated in the cytoplasm of pretangle neurons in varying size granules, which grew into large aggregates co-existing with the progressive development of neurofibrillary tangles. Accumulation of p-MEK1/2 and p-ERK1/2 was found in cases with stages I-III neurofibrillary degeneration, which were devoid of amyloid deposition. These data provide direct in situ evidence consistent with the possible involvement of MAP kinase pathway in the hyperphosphorylation of tau and the presence of this lesion before deposition of beta-amyloid in AD. PMID:12531514

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. Insulin-like growth factor 1 receptor and p38 mitogen-activated protein kinase signals inversely regulate signal transducer and activator of transcription 3 activity to control human dental pulp stem cell quiescence, propagation, and differentiation.

    PubMed

    Vandomme, Jerome; Touil, Yasmine; Ostyn, Pauline; Olejnik, Cecile; Flamenco, Pilar; El Machhour, Raja; Segard, Pascaline; Masselot, Bernadette; Bailliez, Yves; Formstecher, Pierre; Polakowska, Renata

    2014-04-15

    Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Y(low) stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration. PMID:24266654

  18. Ghrelin protects against depleted uranium-induced apoptosis of MC3T3-E1 cells through oxidative stress-mediated p38-mitogen-activated protein kinase pathway.

    PubMed

    Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing; Wang, Weidong; Li, Rong

    2016-01-01

    Depleted uranium (DU) mainly accumulates in the bone over the long term. Osteoblast cells are responsible for the formation of bone, and they are sensitive to DU damage. However, studies investigating methods of reducing DU damage in osteoblasts are rarely reported. Ghrelin is a stomach hormone that stimulates growth hormones released from the hypothalamic-pituitary axis, and it is believed to play an important physiological role in bone metabolism. This study evaluates the impact of ghrelin on DU-induced apoptosis of the osteoblast MC3T3-E1 and investigates its underlying mechanisms. The results show that ghrelin relieved the intracellular oxidative stress induced by DU, eliminated reactive oxygen species (ROS) and reduced lipid peroxidation by increasing intracellular GSH levels; in addition, ghrelin effectively suppressed apoptosis, enhanced mitochondrial membrane potential, and inhibited cytochrome c release and caspase-3 activation after DU exposure. Moreover, ghrelin significantly reduced the expression of DU-induced phosphorylated p38-mitogen-activated protein kinase (MAPK). A specific inhibitor (SB203580) or specific siRNA of p38-MAPK could significantly suppress DU-induced apoptosis and related signals, whereas ROS production was not affected. In addition, ghrelin receptor inhibition could reduce the anti-apoptosis effect of ghrelin on DU and reverse the effect of ghrelin on intracellular ROS and p38-MAPK after DU exposure. These results suggest that ghrelin can suppress DU-induced apoptosis of MC3T3-E1 cells, reduce DU-induced oxidative stress by interacting with its receptor, and inhibit downstream p38-MAPK activation, thereby suppressing the mitochondrial-dependent apoptosis pathway. PMID:26529667

  19. Insulin-Like Growth Factor 1 Receptor and p38 Mitogen-Activated Protein Kinase Signals Inversely Regulate Signal Transducer and Activator of Transcription 3 Activity to Control Human Dental Pulp Stem Cell Quiescence, Propagation, and Differentiation

    PubMed Central

    Vandomme, Jerome; Touil, Yasmine; Ostyn, Pauline; Olejnik, Cecile; Flamenco, Pilar; El Machhour, Raja; Segard, Pascaline; Masselot, Bernadette; Bailliez, Yves; Formstecher, Pierre

    2014-01-01

    Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Ylow stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration. PMID:24266654

  20. Blocking the mitogen activated protein kinase-p38 pathway is associated with increase expression of nitric oxide synthase and higher production of nitric oxide by bovine macrophages infected with Mycobacterium avium subsp paratuberculosis.

    PubMed

    Souza, Cleverson D

    2015-03-15

    This study evaluated the role of the mitogen-activated protein kinase (MAPK)-p38 pathway in the nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by bovine monocyte-derived macrophages ingesting Mycobacterium avium subsp. paratuberculosis (MAP) organisms in vitro. Bovine monocyte-derived macrophages were incubated with MAP organisms with or without a specific inhibitor of the MAPKp38 pathway and activation of the MAPKp38, interleukin - (IL) IL-10, IL-12, iNOS mRNA expression and NO production were evaluated. Incubation of macrophages with MAP organisms activates the MAPKp38 pathway at early time points post infection. Chemically inhibition of MAPKp38 before incubation of bovine macrophages with MAP resulted in increased expression of IL-12 mRNA at 2, 6 and 24h, decreased expression of IL-10 mRNA at 2, 6 and 24h and increased expression of iNOS mRNA at 2 and 6h. Nitric oxide was evaluated to indirectly determine the effects of MAPKp38 pathway on the anti-microbial activity of bovine macrophages. Incubation of bovine macrophages with MAP resulted in modest increased production of NO at 4 and 6h post infection. Pretreatment of bovine macrophages with the MAPKp38 inhibitor SB203580 before addition of MAP organisms resulted in increased production of NO at 2, 4, 6 and 24h post infection. This study expanded our knowledge of the importance of the MAPKp38 pathway in limiting an appropriate macrophage response to MAP and suggested how activation of MAPKp38 pathway may be a target of this organism to disrupt earlier antimicrobial mechanisms of macrophages. These findings raises the interesting possibility that the cellular manipulation of MAPKp38 may be useful in designing novel vaccines against MAP. PMID:25700780

  1. Chronic brain inflammation causes a reduction in GluN2A and GluN2B subunits of NMDA receptors and an increase in the phosphorylation of mitogen-activated protein kinases in the hippocampus

    PubMed Central

    2014-01-01

    Neuroinflammation plays a key role in the initiation and progression of neurodegeneration in Alzheimer’s disease (AD). Chronic neuroinflammation results in diminished synaptic plasticity and loss of GluN1 N-methyl-D-aspartate (NMDA) receptors in the hippocampus, leading to the cognitive deficits that are the most common symptoms of AD. Therefore, it is suggested that chronic inflammation may alter expression levels of GluN2A and GluN2B subunits of NMDA receptors and associated intracellular signalling. Chronic neuroinflammation was induced by chronic infusion of lipopolysaccharide (LPS) into the fourth ventricle in Fischer-344 rats. The status of hippocampus-dependent memory was evaluated in control rats and rats chronically infused with LPS. Microglial activation in the hippocampus was examined using immunohistochemical staining. Western blot analysis was used to measure membrane levels of GluN2A and GluN2B subunits of NMDA receptors and mitogen-activated protein kinase (MAPK) in the hippocampi of these rats, and immunofluorescent double labeling was used to assess the cellular location of MAPK. Microglial activation was observed in the hippocampi of rats that showed memory impairments with chronic LPS infusion. Chronic LPS infusion reduced the levels of GluN2A and GluN2B and increased the levels of phosphorylated MAPKs in the hippocampus. MAPK-positive immunoreactivity was observed mostly in the neurons and also in non-neuronal cells. Reductions in GluN2A and GluN2B subunits of NMDA receptors coupled with altered MAPK signaling, in response to inflammatory stimuli may be related to the cognitive deficits observed in AD. PMID:24761931

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

    PubMed

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

    2015-01-01

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

  3. Tumor Necrosis Factor-α– and Interleukin-1β–Dependent Matrix Metalloproteinase-3 Expression in Nucleus Pulposus Cells Requires Cooperative Signaling via Syndecan 4 and Mitogen-Activated Protein Kinase–NF-κB Axis

    PubMed Central

    Wang, Xin; Wang, Hua; Yang, Hao; Li, Jun; Cai, Qiqing; Shapiro, Irving M.; Risbud, Makarand V.

    2015-01-01

    Matrix metalloproteinase-3 (MMP-3) plays an important role in intervertebral disc degeneration, a ubiquitous condition closely linked to low back pain and disability. Elevated expression of syndecan 4, a cell surface heparan sulfate proteoglycan, actively controls disc matrix catabolism. However, the relationship between MMP-3 expression and syndecan 4 in the context of inflammatory disc disease has not been clearly defined. We investigated the mechanisms by which cytokines control MMP-3 expression in rat and human nucleus pulposus cells. Cytokine treatment increased MMP-3 expression and promoter activity. Stable silencing of syndecan 4 blocked cytokine-mediated MMP-3 expression; more important, syndecan 4 did not mediate its effects through NF-κB or mitogen-activated protein kinase (MAPK) pathways. However, treatment with MAPK and NF-κB inhibitors resulted in partial blocking of the inductive effect of cytokines on MMP-3 expression. Loss-of-function studies confirmed that NF-κB, p38α/β2/γ/δ, and extracellular signal–regulated kinase (ERK) 2, but not ERK1, contributed to cytokine-dependent induction of MMP3 promoter activity. Similarly, inhibitor treatments, lentiviral short hairpin-p65, and short hairpin-IκB kinase β significantly decreased cytokine-dependent up-regulation in MMP-3 expression. Finally, we show that transforming growth factor-β can block the up-regulation of MMP-3 induced by tumor necrosis factor (TNF)-α by counteracting the NF-κB pathway and syndecan 4 expression. Taken together, our results suggest that cooperative signaling through syndecan 4 and the TNF receptor 1–MAPK–NF-κB axis is required for TNF-α–dependent expression of MMP-3 in nucleus pulposus cells. Controlling these pathways may slow the progression of intervertebral disc degeneration and matrix catabolism. PMID:25063530

  4. 17,18-epoxyeicosatetraenoic acid targets PPARγ and p38 mitogen-activated protein kinase to mediate its anti-inflammatory effects in the lung: role of soluble epoxide hydrolase.

    PubMed

    Morin, Caroline; Sirois, Marco; Echavé, Vincent; Albadine, Roula; Rousseau, Eric

    2010-11-01

    This study sought to assess putative pathways involved in the anti-inflammatory effects of 17,18-epoxyeicosatetraenoic acid (17,18-EpETE), as measured by a decrease in the contractile reactivity and Ca(2+) sensitivity of TNF-α-pretreated human bronchi. Tension measurements performed in the presence of 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a soluble epoxide hydrolase (sEH)-specific inhibitor, demonstrated that 17,18-EpETE reduced the reactivity of TNF-α-pretreated tissues. The overexpression of sEH detected in patients with asthma and TNF-α-treated bronchi contributed to the maintenance of hyperresponsiveness in our models, which involved intracellular proinflammatory cascades. The inhibition of peroxisome proliferator-activated receptor (PPAR)γ by GW9662 abolished 17,18-EpETE + AUDA-mediated anti-inflammatory effects by inducing IκBα degradation and cytokine synthesis, indicating that PPARγ is a molecular target of epoxy-eicosanoids. Western blot analysis revealed that 17,18-EpETE pretreatment reversed the phosphorylation of p38 mitogen-activated protein kinase (p38-MAPK) induced by TNF-α in human bronchi. The Ca(2+) sensitivity of human bronchial explants was also quantified on β-escin permeabilized preparations. The presence of SB203580, a p38-MAPK inhibitor, reversed the effect induced by epoxy-eicosanoid in the presence of AUDA on TNF-α-triggered Ca(2+) hypersensitivity by increasing the phosphorylation level of PKC Potentiated Inhibitor Protein-17 (CPI-17) regulatory protein. Moreover, PPARγ ligands, such as rosiglitazone and 17,18-EpETE, decreased the expression of CPI-17, both at the mRNA and protein levels, whereas this effect was countered by GW9662 treatment in TNF-α-treated bronchi. These results demonstrate that 17,18-EpETE is a potent regulator of human lung inflammation and concomitant hyperresponsiveness, and may represent a valuable asset against critical inflammatory bronchial disorder. PMID:20008283

  5. Angiotensin II-Induced Migration of Vascular Smooth Muscle Cells Is Mediated by p38 Mitogen-Activated Protein Kinase-Activated c-Src Through Spleen Tyrosine Kinase and Epidermal Growth Factor Receptor Transactivation

    PubMed Central

    Mugabe, Benon E.; Yaghini, Fariborz A.; Song, Chi Young; Buharalioglu, Cuneyt K.; Waters, Christopher M.

    2010-01-01

    Angiotensin II (Ang II) stimulates protein synthesis by activating spleen tyrosine kinase (Syk) and DNA synthesis through epidermal growth factor receptor (EGFR) transactivation in vascular smooth muscle cells (VSMCs). This study was conducted to determine whether Syk mediates Ang II-induced migration of aortic VSMCs using a scratch wound approach. Treatment with Ang II (200 nM) for 24 h increased VSMC migration by 1.56 ± 0.14-fold. Ang II-induced VSMC migration and Syk phosphorylation as determined by Western blot analysis were minimized by the Syk inhibitor piceatannol (10 μM) and by transfecting VSMCs with dominant-negative but not wild-type Syk plasmid. Ang II-induced VSMC migration and Syk phosphorylation were attenuated by inhibitors of c-Src [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2)], p38 mitogen-activated protein kinase (MAPK) [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190)], and extracellular signal-regulated kinase (ERK) 1/2 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126)]. SB202190 attenuated p38 MAPK and c-Src but not ERK1/2 phosphorylation, indicating that p38 MAPK acts upstream of c-Src and Syk. The c-Src inhibitor PP2 attenuated Syk and ERK1/2 phosphorylation, suggesting that c-Src acts upstream of Syk and ERK1/2. Ang II- and epidermal growth factor (EGF)-induced VSMC migration and EGFR phosphorylation were inhibited by the EGFR blocker 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) (2 μM). Neither the Syk inhibitor piceatannol nor the dominant-negative Syk mutant altered EGF-induced cell migration or Ang II- and EGF-induced EGFR phosphorylation. The c-Src inhibitor PP2 diminished EGF-induced VSMC migration and EGFR, ERK1/2, and p38 MAPK phosphorylation. The ERK1/2 inhibitor U0126 (10 μM) attenuated EGF-induced cell migration and ERK1/2 but not EGFR phosphorylation. These data suggest that Ang II stimulates VSMC migration via p38 MAPK-activated c-Src through

  6. Angiotensin II-induced migration of vascular smooth muscle cells is mediated by p38 mitogen-activated protein kinase-activated c-Src through spleen tyrosine kinase and epidermal growth factor receptor transactivation.

    PubMed

    Mugabe, Benon E; Yaghini, Fariborz A; Song, Chi Young; Buharalioglu, Cuneyt K; Waters, Christopher M; Malik, Kafait U

    2010-01-01

    Angiotensin II (Ang II) stimulates protein synthesis by activating spleen tyrosine kinase (Syk) and DNA synthesis through epidermal growth factor receptor (EGFR) transactivation in vascular smooth muscle cells (VSMCs). This study was conducted to determine whether Syk mediates Ang II-induced migration of aortic VSMCs using a scratch wound approach. Treatment with Ang II (200 nM) for 24 h increased VSMC migration by 1.56 +/- 0.14-fold. Ang II-induced VSMC migration and Syk phosphorylation as determined by Western blot analysis were minimized by the Syk inhibitor piceatannol (10 microM) and by transfecting VSMCs with dominant-negative but not wild-type Syk plasmid. Ang II-induced VSMC migration and Syk phosphorylation were attenuated by inhibitors of c-Src [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2)], p38 mitogen-activated protein kinase (MAPK) [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190)], and extracellular signal-regulated kinase (ERK) 1/2 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126)]. SB202190 attenuated p38 MAPK and c-Src but not ERK1/2 phosphorylation, indicating that p38 MAPK acts upstream of c-Src and Syk. The c-Src inhibitor PP2 attenuated Syk and ERK1/2 phosphorylation, suggesting that c-Src acts upstream of Syk and ERK1/2. Ang II- and epidermal growth factor (EGF)-induced VSMC migration and EGFR phosphorylation were inhibited by the EGFR blocker 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) (2 microM). Neither the Syk inhibitor piceatannol nor the dominant-negative Syk mutant altered EGF-induced cell migration or Ang II- and EGF-induced EGFR phosphorylation. The c-Src inhibitor PP2 diminished EGF-induced VSMC migration and EGFR, ERK1/2, and p38 MAPK phosphorylation. The ERK1/2 inhibitor U0126 (10 microM) attenuated EGF-induced cell migration and ERK1/2 but not EGFR phosphorylation. These data suggest that Ang II stimulates VSMC migration via p38 MAPK-activated c

  7. Anti-hepatitis B virus effect of matrine-type alkaloid and involvement of p38 mitogen-activated protein kinase and tumor necrosis factor receptor-associated factor 6.

    PubMed

    Chen, Jia-Xin; Shen, Hong-Hui; Niu, Ming; Guo, Yu-Ming; Liu, Xiao-Qiong; Han, Yan-Zhong; Zhang, Ya-Ming; Zhao, Yan-Ling; Bai, Bing-Ke; Zhou, Wen-Jun; Xiao, Xiao-He

    2016-04-01

    The matrine-type alkaloid, oxymatrine inhibits hepatitis B virus (HBV) replication but very little is known about these effects in other matrine-type alkaloids, including sophoridine and sophocarpine. Therefore, we compared the in vitro anti-HBV effects of matrine, oxymatrine, sophocarpine, and sophoridine by treating an HBV-transfected cell line (HepG2.2.15) with 0.4-1.6mM of the compounds for 24 or 72h. The levels of the HBV surface antigen (HBsAg) and e antigen (HBeAg) in the culture medium, as well as the intracellular and extracellular HBV DNA levels, were determined. Metabolomic analysis and detection of the mRNA level of p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor receptor-associated factor (TRAF) 6, extracellular signal-regulated kinase (ERK) 1, NOD-like receptor family pyrin domain containing 10 (NLRP10), and caspase-1 were conducted in sophoridine-treated HepG2.2.15 cells. HepG2.2.15 cell exposure to 0.4-1.6mM sophocarpine or sophoridine for 24h reduced the HBsAg level of the medium more effectively than exposure to matrine and oxymatrine did, and reduced the HBeAg levels more effectively than these compounds did at 1.6mM. Sophoridine (0.4-1.6mM) reduced the cell medium HBV DNA levels more than the same concentrations of matrine, oxymatrine, or sophocarpine did. After 72h, 0.4 and 0.8mM sophoridine reduced HBsAg and intracellular HBV DNA levels more potently than matrine, oxymatrine, or sophocarpine did. Furthermore, sophoridine (0.8mM) potently reduced the cell medium HBeAg levels while the metabolomic analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine for 72h exhibited reduced cycloleucine and phytosphingosine levels. In addition, the mRNA expression analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine showed reduced levels of p38 MAPK, TRAF6, ERK1, NLRP10, and caspase-1. Sophoridine produced more potent anti-HBV effects than matrine, oxymatrine, and sophocarpine did. These effects may be related

  8. Dibenzylbutyrolactone lignans from Forsythia koreana fruits attenuate lipopolysaccharide-induced inducible nitric oxide synthetase and cyclooxygenase-2 expressions through activation of nuclear factor-κb and mitogen-activated protein kinase in RAW264.7 cells.

    PubMed

    Lee, Ji Yun; Cho, Bong Jae; Park, Tae Wook; Park, Byoung Eun; Kim, Soo Jung; Sim, Sang Soo; Kim, Chang Jong

    2010-01-01

    Previously, we reported that dibenzylbutyrolactone lignans (DBLLs) from the fruit of Forsythia koreana NAKAI (Oleaceae) has anti-inflammatory, antioxidant, and anti-asthmatic effects. In this study, to clarify the anti-inflammatory mechanisms of DBLL, we evaluated the effects of DBLLs on lipopolysaccharide-stimulated inducible nitric oxide synthetase (iNOS) and cyclooxygenase-2 (COX-2) expressions, nitric oxide (NO) and prostaglandin E(2) (PGE(2)) productions, nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) activations, inhibitor of κB (IκB) and inhibitor of κB kinase (IKK) phosphorylations in cytosolic proteins, and cytotoxicity in Raw264.7 cells. DBLLs potently suppressed both the enzyme expression and DNA-binding activity of NF-κB. Arctiin, arctigenin (1.0 µM) and matairesinol (10 µM) inhibited the expression of iNOS by 37.71±2.86%, 32.51±4.28%, and 27.44±2.65%, respectively, and arctiin, arctigenin (0.1 µM) and matairesinol (1.0 µM) inhibited COX-2 expression by 37.93±7.81%, 26.70±4.61% and 29.37±5.21%, respectively. The inhibitory effects of DBLLs on NO and PGE(2) productions were the same patterns as those seen for the reductions in iNOS and COX-2 expression, respectively. Arctiin, arctigenin (1.0 µM) and matairesinol (10 µM) significantly (p<0.05) inhibited NF-κB DNA binding by 44.85±6.67%, 44.16±6.61%, and 44.79±5.62%, respectively, and arctiin (0.1 µM) and arctigenin (1.0 µM) significantly (p<0.05) inhibited the phosphorylation of IκB by 20.58±3.86% and 25.99±6.18%, respectively. Furthermore, arctiin, matairesinol (1.0 µM) and arctigenin (10 µM) inhibited the phosphorylation of IKK by 38.80±6.64%, 38.33±6.65%, and 38.57±8.14%, respectively. In addition, DBLLs potently inhibited the lipopolysaccharide (LPS)-induced activation of MAPKs (SAPK/c-Jun NH(2)-terminal kinase (JNK), p38, and extracellular signal receptor-activated kinase (ERK)1/2). Overall, arctiin was the most effective; its effect was nearly

  9. 3′, 5′-Cyclic Adenosine 5′-Monophosphate Response Element-Dependent Transcriptional Regulation of the Secretogranin II Gene Promoter Depends on Gonadotropin-Releasing Hormone-Induced Mitogen-Activated Protein Kinase Activation and the Transactivator Activating Transcription Factor 3

    PubMed Central

    Xie, Jianjun; Roberson, Mark S.

    2008-01-01

    Previous studies demonstrated that GnRH-induced secretogranin II (SgII) promoter regulation required a consensus cAMP response element (CRE) and protein kinase A/CRE binding protein. The present studies examined the role of additional components of the GnRH signaling network on SgII promoter activity with particular attention devoted to CRE-dependent gene regulation. Disruption of the SgII CRE by mutagenesis resulted in inhibition of GnRH agonist (GnRHa) induction of this promoter in αT3-1 cells. Pharmacological and dominant-negative inhibition of the ERK and c-Jun N-terminal kinase (JNK) signaling pathways revealed that GnRHa-induced SgII promoter activity required functional JNK and ERK modules. Combined inhibition of both pathways nearly abolished GnRHa-induced SgII promoter activity. Specific induction of the ERK cascade alone using overexpression of Raf-CAAX was not sufficient to activate the SgII gene promoter. In contrast, overexpression of the catalytic domain of the more pleiotropic MAPK activator, MAPK/ERK kinase-1, was sufficient to induce SgII promoter activity. The effect(s) of mitogen-activated protein/ERK kinase-1 on SgII promoter activity was CRE dependent and was reversed by the combined pharmacological inhibition of both JNK and ERK modules. CRE DNA binding studies demonstrated the recruitment of activating transcription factor (ATF)-3 and c-Jun to the CRE after administration of GnRHa to αT3-1 cells. Specific small interfering RNA knockdown of ATF3 reduced ATF3 DNA binding and the effect of GnRHa on the SgII promoter. These studies support the conclusion that MAPK signaling and ATF3 action are essential for full SgII promoter activation by GnRHa through a canonical CRE. Moreover, we suggest that within the GnRH signaling network, CRE-dependent gene regulation in general may be mediated primarily through the immediate early response gene ATF3. PMID:17962349

  10. Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells

    PubMed Central

    Shen, Miaoqing; Bunaciu, Rodica P.; Congleton, Johanna; Jensen, Holly A.; Sayam, Lavanya G.; Varner, Jeffrey D.; Yen, Andrew

    2014-01-01

    All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation. PMID:21740303

  11. Knockdown of Sec8 enhances the binding affinity of c-Jun N-terminal kinase (JNK)-interacting protein 4 for mitogen-activated protein kinase kinase 4 (MKK4) and suppresses the phosphorylation of MKK4, p38, and JNK, thereby inhibiting apoptosis.

    PubMed

    Tanaka, Toshiaki; Iino, Mitsuyoshi; Goto, Kaoru

    2014-12-01

    The exocyst complex, also called the Sec6/8 complex, is important for targeting exocytic vesicles to specific docking sites on the plasma membrane in yeast and mammalian cells. In addition to these original findings, recent results of studies suggest that Sec8 is also involved in oncogenesis, although the functional implications of Sec8 in cancer cells are not well understood. c-Jun N-terminal kinase-interacting protein 4 (JIP4) is a scaffold protein that plays a crucial role in the regulation of mitogen-activated protein kinase (MAPK) signaling cascades. The present study examined how Sec8 is involved in JIP4-mediated MAPK signaling under apoptotic conditions. It was found that Sec8 binds to and regulates JIP4, and that knockdown of Sec8 enhances the binding of JIP4 to MAPK kinase 4, thereby decreasing the phosphorylation of MAPK kinase 4, JNK, and p38. These results raise the possibility that Sec8 serves as an important regulator of MAPK signaling cascades. PMID:25244576

  12. Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

    PubMed

    Liu, Ying; Li, Yueqin; Zhang, Di; Liu, Jiali; Gou, Kemian; Cui, Sheng

    2015-05-01

    The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the fo