p38 mitogen-activated protein kinase (p38MAPK) upregulates catalase levels in response to low dose H2O2 treatment through enhancement of mRNA stability.

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Sen, Prosenjit; Chakraborty, Prabir Kumar; Raha, Sanghamitra

2005-08-15

V79 fibroblasts were repetitively stressed through multiple exposures to a low dose (30 microM) H2O2 in culture for 4 weeks. Catalase activity, protein levels and mRNA levels increased markedly (5-6-fold) during this time and these augmentations were inhibited by the simultaneous presence of SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38MAPK). p38MAPK became dually phosphorylated and ATF-2, a p38MAPK substrate also became increasingly phosphorylated over the repetitive stress period. Short interfering RNA that induced effective silencing of p38MAPK, was used to silence p38MAPK in V79 fibroblasts. Silencing of p38MAPK drastically hindered the elevation in catalase (protein and mRNA) levels observed after a single low dose (50 microM) of H2O. The rise in catalase mRNA levels induced by low concentration (single and multiple dose) H2O2 treatment was established to be unconnected with transcriptional upregulation but was brought forth primarily by an enhancement in catalase mRNA stability through the action of p38MAPK. Therefore, our data strongly indicate that activation of p38MAPK is a key controlling step in the upregulation of catalase levels by low dose H2O2 treatment.

Stimulation of p38 (HOG1) kinase pathway by ionizing radiation results in downstream modulation of ATF/CREB transcription factor activity in NIH-3T3 cells

International Nuclear Information System (INIS)

Stevenson, Mary Ann; Yao Jin

1997-01-01

Purpose/Objective:p38 kinase, a member of the MAP kinase family, is activated in response to stresses such as high osmolarity and UV irradiation as well exposure to cytokines such as IL1β and TNFα. The kinase is part of a signal transduction pathway that leads from receptor activation through a three kinase cascade resulting in the activation of p38. p38 activation then leads to the phosphorylation of target proteins that include transcription factors such as nuclear factor of interleukin 6 and members of the activating transcription factor (ATF) family, and in addition, the stress protein, HSP27, via activation of MAPKAP2 kinase. In the present report, we have investigated the potential role of p38 in the response of NIH-3T3 cells to ionizing radiation. Materials and Methods:NIH-3T3 cells were grown to confluence in DMEM+10%CS and then serum deprived for 24 hours in DMEM+0.1%CS. Radiation exposures were delivered using a Philips RT250 (250Kvp X-ray tube). Activated forms of p38 kinase and ATF/CREB transcription factors were identified using immunoblotting techniques employing activation specific antibodies raised against the phosphorylated forms of the kinases/transcription factors. Kinase activity was directly measured using immunokinase assays. DNA binding of transcription factors to their respective consensus sequences was assayed by EMSA. Results:We found that p38 becomes rapidly phosphorylated and activated by exposure to ionizing radiation. Significantly, p38 is activated to a similar degree and with a similar time course by serum derpviation and entry of cells into a non-proliferating G 0 state, suggesting a causal role for p38 in quiescence. Phosphorylation of p38 directly correlated with phosphorylation and activation of ATF/CREB family members as well as DNA binding by these activated factors. Conclusion:Activation of p38 kinase and downstream transcription factors may play an important role in the response of cells to ionizing radiation. We are

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

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Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

Human p38δ MAP kinase mediates UV irradiation induced up-regulation of the gene expression of chemokine BRAK/CXCL14

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

2010-01-01

The mitogen-activated protein kinase (MAPK) family comprises ERK, JNK, p38 and ERK5 (big-MAPK, BMK1). UV irradiation of squamous cell carcinoma cells induced up-regulation of gene expression of chemokine BRAK/CXCL14, stimulated p38 phosphorylation, and down-regulated the phosphorylation of ERK. Human p38 MAPKs exist in 4 isoforms: p38α, β, γ and δ. The UV stimulation of p38 phosphorylation was not inhibited by the presence of SB203580 or PD169316, inhibitors of p38α and β, suggesting p38 phosphorylation was not dependent on these 2 isoforms and that p38γ and/or δ was responsible for the phosphorylation. In fact, inhibition of each of these 4 p38 isoforms by the introduction of short hairpin (sh) RNAs for respective isoforms revealed that only shRNA for p38δ attenuated the UV-induced up-regulation of BRAK/CXCL14 gene expression. In addition, over-expression of p38 isoforms in the cells showed the association of p38δ with ERK1 and 2, concomitant with down-regulation of ERK phosphorylation. The usage of p38δ isoform by UV irradiation is not merely due to the abundance of this p38 isoform in the cells. Because serum deprivation of the cells also induced an increase in BRAK/CXCL14 gene expression, and in this case p38α and/or β isoform is responsible for up-regulation of BRAK/CXCL14 gene expression. Taken together, the data indicate that the respective stress-dependent action of p38 isoforms is responsible for the up-regulation of the gene expression of the chemokine BRAK/CXCL14.

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

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

2008-07-01

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

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

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

2009-07-24

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

Low intensity shear stress increases endothelial ELR+ CXC chemokine production via a focal adhesion kinase-p38{beta} MAPK-NF-{kappa}B pathway.

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Shaik, Sadiq S; Soltau, Thomas D; Chaturvedi, Gaurav; Totapally, Balagangadhar; Hagood, James S; Andrews, William W; Athar, Mohammad; Voitenok, Nikolai N; Killingsworth, Cheryl R; Patel, Rakesh P; Fallon, Michael B; Maheshwari, Akhil

2009-02-27

CXC chemokines with a glutamate-leucine-arginine (ELR) tripeptide motif (ELR(+) CXC chemokines) play an important role in leukocyte trafficking into the tissues. For reasons that are not well elucidated, circulating leukocytes are recruited into the tissues mainly in small vessels such as capillaries and venules. Because ELR(+) CXC chemokines are important mediators of endothelial-leukocyte interaction, we compared chemokine expression by microvascular and aortic endothelium to investigate whether differences in chemokine expression by various endothelial types could, at least partially, explain the microvascular localization of endothelial-leukocyte interaction. Both in vitro and in vivo models indicate that ELR(+) CXC chemokine expression is higher in microvascular endothelium than in aortic endothelial cells. These differences can be explained on the basis of the preferential activation of endothelial chemokine production by low intensity shear stress. Low shear activated endothelial ELR(+) CXC chemokine production via cell surface heparan sulfates, beta(3)-integrins, focal adhesion kinase, the mitogen-activated protein kinase p38beta, mitogen- and stress-associated protein kinase-1, and the transcription factor.

Mitochondrial p38β and manganese superoxide dismutase interaction mediated by estrogen in cardiomyocytes.

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Han Liu

Full Text Available While etiology behind the observed acceleration of ischemic heart disease in postmenopausal women is poorly understood, collective scientific data suggest cardioprotective effects of the endogenous female sex hormone, estrogen. We have previously shown that 17β-estradiol (E2 protects cardiomyocytes exposed to hypoxia-reoxygenation (H/R by inhibiting p38α - p53 signaling in apoptosis and activating pro-survival p38β mitogen activated protein kinase (p38β MAPK, leading to suppression of reactive oxygen species (ROS post H/R. However, little is known about the mechanism behind the antioxidant actions of E2-dependent p38β. The aim of this study is to determine whether the cytoprotection by estrogen involves regulation of manganese superoxide dismutase (MnSOD, a major mitochondrial ROS scavenging enzyme, via cardiac p38β.We identified mitochondrial p38β by immunocytochemistry and by immunoblotting in mitochondria isolated from neonatal cardiomyocytes of Sprague-Dawley rats. E2 facilitated the mitochondrial localization of the active form of the kinase, phosphorylated p38β (p-p38β. E2 also reduced the H/R-induced mitochondrial membrane potential decline, augmented the MnSOD activity and suppressed anion superoxide generation, while the dismutase protein expression remained unaltered. Co-immunoprecipitation studies showed physical association between MnSOD and p38β. p38β phosphorylated MnSOD in an E2-dependent manner in in-vitro kinase assays.This work demonstrates for the first time a mitochondrial pool of active p38β and E2-mediated phosphorylation of MnSOD by the kinase. The results shed light on the mechanism behind the cytoprotective actions of E2 in cardiomyocytes under oxidative stress.

Functional Roles of p38 Mitogen-Activated Protein Kinase in Macrophage-Mediated Inflammatory Responses

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Yanyan Yang

2014-01-01

Full Text Available Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α and cyclooxygenase-2 (COX-2. p38 MAPK signaling plays an essential role in regulating cellular processes, especially inflammation. In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.

A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity.

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Kim, Dennis H; Feinbaum, Rhonda; Alloing, Geneviève; Emerson, Fred E; Garsin, Danielle A; Inoue, Hideki; Tanaka-Hino, Miho; Hisamoto, Naoki; Matsumoto, Kunihiro; Tan, Man-Wah; Ausubel, Frederick M

2002-07-26

A genetic screen for Caenorhabditis elegans mutants with enhanced susceptibility to killing by Pseudomonas aeruginosa led to the identification of two genes required for pathogen resistance: sek-1, which encodes a mitogen-activated protein (MAP) kinase kinase, and nsy-1, which encodes a MAP kinase kinase kinase. RNA interference assays and biochemical analysis established that a p38 ortholog, pmk-1, functions as the downstream MAP kinase required for pathogen defense. These data suggest that this MAP kinase signaling cassette represents an ancient feature of innate immune responses in evolutionarily diverse species.

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

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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. Copyright © 2015 Elsevier Ltd. All rights reserved.

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

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Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

2013-06-10

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

Tumor Necrosis Factor-α and Apoptosis Signal-Regulating Kinase 1 Control Reactive Oxygen Species Release, Mitochondrial Autophagy and C-Jun N-Terminal Kinase/P38 Phosphorylation During Necrotizing Enterocolitis

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Naira Baregamian

2009-01-01

Full Text Available Background: Oxidative stress and inflammation may contribute to the disruption of the protective gut barrier through various mechanisms; mitochondrial dysfunction resulting from inflammatory and oxidative injury may potentially be a significant source of apoptosis during necrotizing enterocolitis (NEC. Tumor necrosis factor (TNFα is thought to generate reactive oxygen species (ROS and activate the apoptosis signal-regulating kinase 1 (ASK1-c-Jun N-terminal kinase (JNK/p38 pathway. Hence, the focus of our study was to examine the effects of TNFα/ROs on mitochondrial function, ASK1-JNK/p38 cascade activation in intestinal epithelial cells during NEC.

Inhibition of Cartilage Acidic Protein 1 Reduces Ultraviolet B Irradiation Induced-Apoptosis through P38 Mitogen-Activated Protein Kinase and Jun Amino-Terminal Kinase Pathways

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Yinghong Ji

2016-11-01

Full Text Available Background/Aims: Ultraviolet B (UVB irradiation can easily induce apoptosis in human lens epithelial cells (HLECs and further lead to various eye diseases including cataract. Here for the first time, we investigated the role of cartilage acidic protein 1 (CRTAC1 gene in UVB irradiation induced-apoptosis in HLECs. Methods: Three groups of HLECs were employed including model group, empty vector group, and CRTAC1 interference group. Results: After UVB irradiation, the percentage of primary apoptotic cells was obviously fewer in CRTAC1 interference group. Meanwhile, inhibition of CRTAC1 also reduced both reactive oxygen species (ROS production and intracellular Ca2+ concentration, but the level of mitochondrial membrane potential (Δψm was increased in HLECs. Further studies indicated that superoxide dismutase (SOD activity and total antioxidative (T-AOC level were significantly increased in CRTAC1-inhibited cells, while the levels of malondialdehyde (MDA and lactate dehydrogenase (LDH were significantly decreased. ELISA analysis of CRTAC1-inhibited cells showed that the concentrations of tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6 were significantly decreased, but the concentration of interleukin-10 (IL-10 was significantly increased. Western blot analyses of eight apoptosis-associated proteins including Bax, Bcl-2, p38, phospho-p38 (p-p38, Jun amino-terminal kinases (JNK1/2, phospho-JNK1/2 (p-JNK1/2, calcium-sensing receptor (CasR, and Ca2+/calmodulin-dependent protein kinase II (CaMKII indicated that the inhibition of CRTAC1 alleviated oxidative stress and inflammation response, inactivated calcium-signaling pathway, p38 and JNK1/2 signal pathways, and eventually reduced UVB irradiation induced-apoptosis in HLECs. Conclusion: These results provided new insights into the mechanism of cataract development, and demonstrated that CRTAC1 could be a potentially novel target for cataract treatment.

Inhibition of Cartilage Acidic Protein 1 Reduces Ultraviolet B Irradiation Induced-Apoptosis through P38 Mitogen-Activated Protein Kinase and Jun Amino-Terminal Kinase Pathways.

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Ji, Yinghong; Rong, Xianfang; Li, Dan; Cai, Lei; Rao, Jun; Lu, Yi

2016-01-01

Ultraviolet B (UVB) irradiation can easily induce apoptosis in human lens epithelial cells (HLECs) and further lead to various eye diseases including cataract. Here for the first time, we investigated the role of cartilage acidic protein 1 (CRTAC1) gene in UVB irradiation induced-apoptosis in HLECs. Three groups of HLECs were employed including model group, empty vector group, and CRTAC1 interference group. After UVB irradiation, the percentage of primary apoptotic cells was obviously fewer in CRTAC1 interference group. Meanwhile, inhibition of CRTAC1 also reduced both reactive oxygen species (ROS) production and intracellular Ca2+ concentration, but the level of mitochondrial membrane potential (Δψm) was increased in HLECs. Further studies indicated that superoxide dismutase (SOD) activity and total antioxidative (T-AOC) level were significantly increased in CRTAC1-inhibited cells, while the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) were significantly decreased. ELISA analysis of CRTAC1-inhibited cells showed that the concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly decreased, but the concentration of interleukin-10 (IL-10) was significantly increased. Western blot analyses of eight apoptosis-associated proteins including Bax, Bcl-2, p38, phospho-p38 (p-p38), Jun amino-terminal kinases (JNK1/2), phospho-JNK1/2 (p-JNK1/2), calcium-sensing receptor (CasR), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) indicated that the inhibition of CRTAC1 alleviated oxidative stress and inflammation response, inactivated calcium-signaling pathway, p38 and JNK1/2 signal pathways, and eventually reduced UVB irradiation induced-apoptosis in HLECs. These results provided new insights into the mechanism of cataract development, and demonstrated that CRTAC1 could be a potentially novel target for cataract treatment. © 2016 The Author(s) Published by S. Karger AG, Basel.

N-3 Polyunsaturated Fatty Acids Decrease the Protein Expression of Soluble Epoxide Hydrolase via Oxidative Stress-Induced P38 Kinase in Rat Endothelial Cells.

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Okada, Takashi; Morino, Katsutaro; Nakagawa, Fumiyuki; Tawa, Masashi; Kondo, Keiko; Sekine, Osamu; Imamura, Takeshi; Okamura, Tomio; Ugi, Satoshi; Maegawa, Hiroshi

2017-06-24

N -3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n -3 PUFAs, increased in n -3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n -3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n -3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n -3 PUFAs may contribute to their cardio-protective effect.

dsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

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Eliane F. Meurs

2012-10-01

Full Text Available The double-stranded RNA-dependent protein kinase PKR plays multiple roles in cells, in response to different stress situations. As a member of the interferon (IFN‑Stimulated Genes, PKR was initially recognized as an actor in the antiviral action of IFN, due to its ability to control translation, through phosphorylation, of the alpha subunit of eukaryotic initiation factor 2 (eIF2a. As such, PKR participates in the generation of stress granules, or autophagy and a number of viruses have designed strategies to inhibit its action. However, PKR deficient mice resist most viral infections, indicating that PKR may play other roles in the cell other than just acting as an antiviral agent. Indeed, PKR regulates several signaling pathways, either as an adapter protein and/or using its kinase activity. Here we review the role of PKR as an eIF2a kinase, its participation in the regulation of the NF-kB, p38MAPK and insulin pathways, and we focus on its role during infection with the hepatitis C virus (HCV. PKR binds the HCV IRES RNA, cooperates with some functions of the HCV core protein and may represent a target for NS5A or E2. Novel data points out for a role of PKR as a pro-HCV agent, both as an adapter protein and as an eIF2a-kinase, and in cooperation with the di-ubiquitin-like protein ISG15. Developing pharmaceutical inhibitors of PKR may help in resolving some viral infections as well as stress-related damages.

HSP90 inhibitors potentiate PGF2α-induced IL-6 synthesis via p38 MAP kinase in osteoblasts.

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Kazuhiko Fujita

Full Text Available Heat shock protein 90 (HSP90 that is ubiquitously expressed in various tissues, is recognized to be a major molecular chaperone. We have previously reported that prostaglandin F2α (PGF2α, a potent bone remodeling mediator, stimulates the synthesis of interleukin-6 (IL-6 through p44/p42 mitogen-activated protein (MAP kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that Rho-kinase acts at a point upstream of p38 MAP kinase. In the present study, we investigated the involvement of HSP90 in the PGF2α-stimulated IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. Geldanamycin, an inhibitor of HSP90, significantly amplified both the PGF2α-stimulated IL-6 release and the mRNA expression levels. In addition, other HSP90 inhibitors, 17-allylamino-17demethoxy-geldanamycin (17-AAG and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG and onalespib, enhanced the PGF2α-stimulated IL-6 release. Geldanamycin, 17-AAG and onalespib markedly strengthened the PGF2α-induced phosphorylation of p38 MAP kinase. Geldanamycin and 17-AAG did not affect the PGF2α-induced phosphorylation of p44/p42 MAP kinase and myosin phosphatase targeting subunit (MYPT-1, a substrate of Rho-kinase, and the protein levels of RhoA and Rho-kinase. In addition, HSP90-siRNA enhanced the PGF2α-induced phosphorylation of p38 MAP kinase. Furthermore, SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by geldanamycin, 17-AAG or 17-DMAG of the PGF2α-stimulated IL-6 release. Our results strongly suggest that HSP90 negatively regulates the PGF2α-stimulated IL-6 synthesis in osteoblasts, and that the effect of HSP90 is exerted through regulating p38 MAP kinase activation.

Molecular Cloning and Characterization of a P38-Like Mitogen-Activated Protein Kinase from Echinococcus granulosus.

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Lü, Guodong; Li, Jing; Zhang, Chuanshan; Li, Liang; Bi, Xiaojuan; Li, Chaowang; Fan, Jinliang; Lu, Xiaomei; Vuitton, Dominique A; Wen, Hao; Lin, Renyong

2016-12-01

Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus . We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus . Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens p38α, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for p38α. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with TGF-β1 effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus , as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human TGF-β1.

p18(Hamlet) mediates different p53-dependent responses to DNA-damage inducing agents.

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Lafarga, Vanesa; Cuadrado, Ana; Nebreda, Angel R

2007-10-01

Cells organize appropriate responses to environmental cues by activating specific signaling networks. Two proteins that play key roles in coordinating stress responses are the kinase p38alpha (MAPK14) and the transcription factor p53 (TP53). Depending on the nature and the extent of the stress-induced damage, cells may respond by arresting the cell cycle or by undergoing cell death, and these responses are usually associated with the phosphorylation of particular substrates by p38alpha as well as the activation of specific target genes by p53. We recently characterized a new p38alpha substrate, named p18(Hamlet) (ZNHIT1), which mediates p53-dependent responses to different genotoxic stresses. Thus, cisplatin or UV light induce stabilization of the p18(Hamlet) protein, which then enhances the ability of p53 to bind to and activate the promoters of pro-apoptotic genes such as NOXA and PUMA leading to apoptosis induction. In a similar way, we report here that p18(Hamlet) can also mediate the cell cycle arrest induced in response to gamma-irradiation, by participating in the p53-dependent upregulation of the cell cycle inhibitor p21(Cip1) (CDKN1A).

Involvement of p38 mitogen-activated protein kinase in acquired gemcitabine-resistant human urothelial carcinoma sublines

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Yu-Ting Kao

2014-07-01

Full Text Available Resistance to chemotherapeutic drugs is one of the major challenges in the treatment of cancer. A better understanding of how resistance arises and what molecular alterations correlate with resistance is the key to developing novel effective therapeutic strategies. To investigate the underlying mechanisms of gemcitabine (Gem resistance and provide possible therapeutic options, three Gem-resistant urothelial carcinoma sublines were established (NG0.6, NG0.8, and NG1.0. These cells were cross-resistant to arabinofuranosyl cytidine and cisplatin, but sensitive to 5-fluorouracil. The resistant cells expressed lower values of [hENT1 × dCK/RRM1 × RRM2] mRNA ratio. Two adenosine triphosphate-binding cassette proteins ABCD1 as well as multidrug resistance protein 1 were elevated. Moreover, cyclin D1, cyclin-dependent kinases 2 and 4 were upregulated, whereas extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK activity were repressed significantly. Administration of p38 MAPK inhibitor significantly reduced the Gem sensitivity in NTUB1 cells, whereas that of an extracellular signal-regulated kinase MAPK inhibitor did not. Furthermore, the Gem-resistant sublines also exhibited higher migration ability. Forced expression of p38 MAPK impaired the cell migration activity and augmented Gem sensitivity in NG1.0 cells. Taken together, these results demonstrate that complex mechanisms were merged in acquiring Gem resistance and provide information that can be important for developing therapeutic targets for treating Gem-resistant tumors.

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

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Yang, Se-Ran; Cho, Sung-Dae; Ahn, Nam-Shik; Jung, Ji-Won; Park, Joon-Suk; Jo, Eun-Hye; Hwang, Jae-Woong; Kim, Sung-Hoon; Lee, Bong-Hee; Kang, Kyung-Sun; Lee, Yong-Soon

2005-01-01

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK. These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis

Phosphorylation of the Grb2- and phosphatidylinositol 3-kinase p85-binding p36/38 by Syk in Lck-negative T cells.

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von Willebrand, M; Williams, S; Tailor, P; Mustelin, T

1998-06-01

Activation of the mitogen-activated protein kinase (MAPK) pathway by the T-cell antigen receptor (TCR) in T cells involves a positive role for phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enabled the TCR to induce a normal activation of the Erk2 MAPK and enhanced transcription of a reporter gene driven by the nuclear factor of activated T cells and AP-1. Because this system allows us to analyse the targets for Syk in receptor-mediated signalling, we examined the role of PI3K in signalling events between the TCR-regulated Syk and the downstream activation of Erk2. We report that inhibition of PI3K by wortmannin or an inhibitory p85 construct, p85deltaiSH2, reduced the TCR-induced Syk-dependent activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same time, expression of Syk resulted in the activation-dependent phosphorylation of three proteins that bound to the src homology 2 (SH2) domains of PI3K p85. The strongest of these bands had an apparent molecular mass of 36-38 kDa on SDS gels, and it was quantitatively removed from the lysates by adsorption to a fusion protein containing the SH2 domain of Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphorylated by Syk or a Syk-regulated kinase, and this protein may provide a link to the recruitment and activation of PI3K, as well as to the Ras-MAPK pathway, in TCR-triggered T cells.

STATE OF JNK AND P38 MAP-KINASE SYSTEM IN BLOOD monon uclea r le ucocytes DUR ING INFLAMMATION

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N. Y. Chasovskih

2009-01-01

Full Text Available Abstract. Pogrammed cell death of peripheral blood mononuclear leucocytes from patients with acute inflammatory diseases (non-nosocomial pneumonia, acute appendicitis was investigated under ex vivo conditions, upon cultivation of the cells with selective inhibitors of JNK (SP600125 and р38 МАРК (ML3403. In vitro addition of SP600125 and ML3403 under oxidative stress conditions prevents increase of annexinpositive mononuclear cells numbers, thus suggesting JNK and р38 МАР-kinases to be involved into oxidative mechanisms of apoptosis deregulation. A role of JNK in IL-8 production by mononuclear leucocytes was revealed in cases of acute inflammation. Regulatory effect of JNK and p38 MAP-kinases can be mediated through activation of redox-sensitive apoptogenic signal transduction systems, as well as due to changes in cellular cytokine-producing function.

Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture.

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Boon Siang Nicholas Tan

Full Text Available Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL cells from mouse embryonic stem (mES cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation.

Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

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Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

2016-01-01

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

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Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L; Hood, Molly M; Lord, John W; Lu, Wei-Ping; Miller, David F; Patt, William C; Smith, Bryan D; Vogeti, Lakshminarayana; Kaufman, Michael D; Petillo, Peter A; Wise, Scott C; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L

2010-10-01

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase. Copyright © 2010 Elsevier Ltd. All rights reserved.

Targeting p38 Mitogen-Activated Protein Kinase Signaling Restores Subventricular Zone Neural Stem Cells and Corrects Neuromotor Deficits in Atm Knockout Mouse

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Kim, Jeesun

2012-01-01

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

p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland.

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Carlsson, Stina K; Gierow, J Peter

2012-03-01

The lacrimal gland (LG) is an exocrine gland important for secretion of the tear film. The kinase p38 has important signal transduction functions, e.g. in gene transcription, but has previously not been known to modulate exocrine secretion. The aim of the current study was to investigate the role of p38 in carbachol (Cch)-induced LG secretion in LG acinar cells in vitro. Western blotting was used to determine the phosphorylation status of p38 and p42/44 and determine expression of p38 isoforms. To determine the effect of p38 inhibition on LG secretion, PD 169316, a general p38 inhibitor, and SB 239063, an inhibitor of p38α and β, were added to the cells prior to secretion measurements. The results revealed activation of p38 mediated by Cch stimulation and inhibition of Cch-induced secretion as a result of p38 inhibition. The inhibition was observed with PD 169316 isoforms, but not with SB 239063. The p38δ isoform was shown to have robust expression both by Western blotting of acinar cells and immunofluorescence of the whole gland. In conclusion, p38 activation mediates secretion in cholinergic stimulation of rabbit LG cells.

Heat Shock Protein 70 Negatively Regulates TGF-β-Stimulated VEGF Synthesis via p38 MAP Kinase in Osteoblasts

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Go Sakai

2017-11-01

Full Text Available Background/Aims: We previously demonstrated that transforming growth factor-β (TGF-β stimulates the synthesis of vascular endothelial growth factor (VEGF through the activation of p38 mitogen-activated protein (MAP kinase in osteoblast-like MC3T3-E1 cells. Heat shock protein70 (HSP70 is a ubiquitously expressed molecular chaperone. In the present study, we investigated the involvement of HSP70 in the TGF-β-stimulated VEGF synthesis and the underlying mechanism in these cells. Methods: Culture MC3T3-E1 cells were stimulated by TGF-β. Released VEGF was measured using an ELISA assay. VEGF mRNA level was quantified by RT-PCR. Phosphorylation of each protein kinase was analyzed by Western blotting. Results: VER-155008 and YM-08, both of HSP70 inhibitors, significantly amplified the TGF-β-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-β was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-β-induced phosphorylation of p38 MAP kinase. The TGF-β-induced phosphorylation of p38 MAP kinase was amplified in HSP70-knockdown cells. SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by these inhibitors of the TGF-β-induced VEGF release. Conclusion: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-β-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

International Nuclear Information System (INIS)

Choi, Cheol-Hee; Lee, Byung-Hoon; Ahn, Sang-Gun; Oh, Seon-Hee

2012-01-01

Highlights: ► MG132 induces the phosphorylation of GSK3β Ser9 and, to a lesser extent, of GSK3β Thr390 . ► MG132 induces dephosphorylation of p70S6K Thr389 and phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 dephosphorylates GSK3β Ser9 and phosphorylates GSK3β Thr390 . ► Inactivation of p38 phosphorylates p70S6K Thr389 and increases the phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser 9 and, to a lesser extent, Thr 390 , the dephosphorylation of p70S6K at Thr 389 , and the phosphorylation of p70S6K at Thr 421 and Ser 424 . The specific p38 inhibitor SB203080 reduced the p-GSK3β Ser9 and autophagy through the phosphorylation of p70S6K Thr389 ; however, it augmented the levels of p-ERK, p-GSK3β Thr390 , and p-70S6K Thr421/Ser424 induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK Ser9 /p70S6K Thr380 and ERK/GSK3β Thr390 /p70S6K Thr421/Ser424 kinase signaling, which is involved in cell survival and cell death.

Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factor-β receptor-mediated ERK and p38 pathways

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Zhang, Feng; Ni, Chunyan; Kong, Desong; Zhang, Xiaoping; Zhu, Xiaojing; Chen, Li; Lu, Yin; Zheng, Shizhong

2012-01-01

Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with accumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fibrosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSC activation stimulated by hydrogen peroxide (H 2 O 2 ), an in vitro model mimicking the oxidative stress in liver fibrogenesis, and to elucidate the possible mechanisms. Our results demonstrated that H 2 O 2 at 5 μM significantly stimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazine dose-dependently suppressed proliferation and induced apoptosis in H 2 O 2 -activated HSCs, and attenuated expression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factor-β receptor (PDGF-βR) expression and blocked the phosphorylation of extracellular regulated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-βR. Further molecular evidence suggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-βR and might be involved in ligustrazine reduction of fibrotic marker gene expression under H 2 O 2 stimulation. Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H 2 O 2 -stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by oxidative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis. Highlights: ► Ligustrazine inhibits oxidative stress-induced HSC activation. ► Ligustrazine reduces fibrotic marker genes

An inhibition of p38 mitogen activated protein kinase delays the platelet storage lesion.

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Andrey Skripchenko

Full Text Available BACKGROUND AND OBJECTIVES: Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK. Another MAPK, extracellular signal-related kinase (ERK, is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors. MATERIALS AND METHODS: A single Trima apheresis platelet unit (n = 12 was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20-24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters. RESULTS: Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties. CONCLUSION: Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca2+]i elevation

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Chou, C.-T.; He Shiping; Jan, C.-R.

2007-01-01

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

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3{beta}

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Choi, Cheol-Hee [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Department of Pharmacology, College of Medicine, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Lee, Byung-Hoon [College of Pharmacy and Multiscreening Center for Drug Development, Seoul National University, Seoul 151-742 (Korea, Republic of); Ahn, Sang-Gun [Department of Pathology, College of Dentistry, Chosun University, Gwangju 501-759 (Korea, Republic of); Oh, Seon-Hee, E-mail: oshccw@hanmail.net [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of)

2012-02-24

Highlights: Black-Right-Pointing-Pointer MG132 induces the phosphorylation of GSK3{beta}{sup Ser9} and, to a lesser extent, of GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer MG132 induces dephosphorylation of p70S6K{sup Thr389} and phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 dephosphorylates GSK3{beta}{sup Ser9} and phosphorylates GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer Inactivation of p38 phosphorylates p70S6K{sup Thr389} and increases the phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3{beta} (GSK3{beta}) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3{beta} at Ser{sup 9} and, to a lesser extent, Thr{sup 390}, the dephosphorylation of p70S6K at Thr{sup 389}, and the phosphorylation of p70S6K at Thr{sup 421} and Ser{sup 424}. The specific p38 inhibitor SB203080 reduced the p-GSK3{beta}{sup Ser9} and autophagy through the phosphorylation of p70S6K{sup Thr389}; however, it augmented the levels of p-ERK, p-GSK3{beta}{sup Thr390}, and p-70S6K{sup Thr421/Ser424} induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our

MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK

KAUST Repository

Zhang, Gen

2013-07-29

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway. © 2013 Zhang et al.

MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK

KAUST Repository

Zhang, Gen; He, Li-Sheng; Wong, Yue Him; Qian, Pei-Yuan

2013-01-01

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway. © 2013 Zhang et al.

Arsenic trioxide inhibits Ewing's sarcoma cell invasiveness by targeting p38(MAPK) and c-Jun N-terminal kinase.

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Zhang, Shuai; Guo, Wei; Ren, Ting-Ting; Lu, Xin-Chang; Tang, Guo-Qing; Zhao, Fu-Long

2012-01-01

Ewing's sarcoma is the second most frequent primary malignant bone tumor, mainly affecting children and young adults. The notorious metastatic capability of this tumor aggravates patient mortality and remains a problem to be overcome. We investigated the effect of arsenic trioxide (As₂O₃) on the metastasis capability of Ewing's sarcoma cells. We performed 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide assays to choose appropriate concentrations of As₂O₃ for the experiments. Migration, invasion, and adhesion assays were performed to assess the effect of As₂O₃ on the metastasis of Ewing's sarcoma. Immunofluorescent staining was used to observe cytoskeleton reorganization in Ewing's sarcoma cells treated with As₂O₃. Changes in matrix metalloproteinase-9 expression and the mitogen-activated protein kinase (MAPK) pathway were investigated using western blot. Inhibitors of p38(MAPK) (sb202190) and c-Jun NH₂-terminal kinase (JNK, sp600125) were used in invasion assays to determine the effect of p38(MAPK) and JNK. We found that As₂O₃ may markedly inhibit the migration and invasion capacity of Ewing's sarcoma cells with structural rearrangements of the actin cytoskeleton. The expressions of matrix metalloproteinase-9, phosphor-p38(MAPK), and phosphor-JNK were suppressed by As₂O₃ treatment in a dose-dependent manner. The inhibitors of p38(MAPK) (sb202190) and JNK (sp600125) enhanced the inhibition induced by As₂O₃, which was counteracted by anisomycin, an activating agent of p38(MAPK) and JNK. Taken together, our results demonstrate that As₂O₃ can inhibit the metastasis capability of RD-ES and A-673 cells and may have new therapeutic value for Ewing's sarcoma.

2,2',4,4'-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-κB

International Nuclear Information System (INIS)

Bezdecny, Steven A.; Karmaus, Peer; Roth, Robert A.; Ganey, Patricia E.

2007-01-01

-mediated increases in superoxide anion were prevented by the NADPH oxidase inhibitor apocynin or the free radical scavenger 4-hydroxy TEMPO, but neither of these inhibitors affected the 2244-TCB-induced changes in COX-2 mRNA levels or 3 H-AA release. Taken together these data suggest that p38 MAP kinase-dependent activation of NF-κB is critical for the 2244-TCB-mediated upregulation of COX-2 mRNA

Detection of protein kinases P38 based on reflectance spectroscopy with n-type porous silicon microcavities for diagnosing hydatidosis hydatid disease

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Lv, Xiaoyi; Lv, Guodong; Jia, Zhenhong; Wang, Jiajia; Mo, Jiaqing

2014-11-01

Detection of protein kinases P38 of Echinococcus granulosus and its homologous antibody have great value for early diagnosis and treatment of hydatidosis hydatid disease. In this experiment, n-type mesoporous silicon microcavities have been successfully fabricated without KOH etching or oxidants treatment that reported in other literature. We observed the changes of the reflectivity spectrum before and after the antigen-antibody reaction by n-type mesoporous silicon microcavities. The binding of protein kinases P38 and its homologous antibody causes red shifts in the reflection spectrum of the sensor, and the red shift was proportional to the protein kinases P38 concentration with linear relationship.

Involvement of a novel p38 mitogen-activated protein kinase in larval metamorphosis of the polychaete Hydroides elegans (Haswell)

KAUST Repository

Wang, Hao

2010-04-19

Hydroides elegans is a common marine fouling organism in most tropical and subtropical waters. The life cycle of H. elegans includes a planktonic larval stage in which swimming larvae normally take 5 days to attain competency to settle. Larval metamorphosis marks the beginning of its benthic life; however, the endogenous molecular mechanisms that regulate metamorphosis remain largely unknown. In this study, a PCR-based suppressive subtractive hybridization (SSH) library was constructed to screen the genes expressed in competent larvae but not in precompetent larvae. Among the transcripts isolated from the library, 21 significantly matched sequences in the GenBank. Many of these isolated transcripts have putative roles in the reactive oxygen species (ROS) signal transduction pathway or in response to ROS stress. A putative novel p38 mitogen-activated protein kinase (MAPK), which was also isolated with SSH screen, was then cloned and characterized. The MAPK inhibitors assay showed that both p38 MAPK inhibitors SB202190 and SB203580 effectively inhibited the biofilm-induced metamorphosis of H. elegans. A cell stressors assay showed that H2O2 effectively induced larval metamorphosis of H. elegans, but the inductivity of H2O2 was also inhibited by both SB inhibitors. The catalase assay showed that the catalase could effetely inhibit H. elegans larvae from responding to inductive biofilm. These results showed that the p38 MAPK-dependent pathway plays critical role in controlling larval metamorphosis of the marine polychaete H. elegans, and the reactive oxygen radicals produced by biofilm could be the cue inducing larval metamorphosis. © 2010 Wiley-Liss, Inc.

p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-uncoupling in obesity.

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Yu, Yi; Rajapakse, Angana G; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

2014-07-18

Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model. Obesity was induced in wild type (WT) and Arg-II-deficient (Arg-II(-/-)) mice on C57BL/6 J background by high-fat diet (HFD, 55% fat) for 14 weeks starting from age of 7 weeks. The entire aortas were isolated and subjected to 1) immunoblotting analysis of the protein level of eNOS, Arg-II and p38mapk activation; 2) arginase activity assay; 3) endothelium-dependent and independent vasomotor responses; 4) en face staining of superoxide anion and NO production with Dihydroethidium and 4,5-Diaminofluorescein Diacetate, respectively, to assess eNOS-uncoupling. To evaluate the role of p38mapk, isolated aortas were treated with p38mapk inhibitor SB203580 (10 μmol/L, 1 h) prior to the analysis. In addition, the role of p38mapk in Arg-II-induced eNOS-uncoupling was investigated in cultured human endothelial cells overexpressing Arg-II in the absence or presence of shRNA against p38mapk. HFD enhanced Arg-II expression/activity and p38mapk activity, which was associated with eNOS-uncoupling as revealed by decreased NO and enhanced L-NAME-inhibitable superoxide in aortas of WT obese mice. In accordance, WT obese mice revealed decreased endothelium-dependent relaxations to acetylcholine despite of higher eNOS protein level, whereas Arg-II(-/-) obese mice were protected from HFD-induced eNOS-uncoupling and endothelial dysfunction, which

Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

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Badrinarayan, Preethi; Sastry, G Narahari

2012-04-01

In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. Copyright © 2012 Elsevier Inc. All rights reserved.

FANCA and FANCC modulate TLR and p38 MAPK-dependent expression of IL-1β in macrophages.

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Garbati, Michael R; Hays, Laura E; Keeble, Winifred; Yates, Jane E; Rathbun, R Keaney; Bagby, Grover C

2013-10-31

Hematopoietic stem and progenitor cells with inactivated Fanconi anemia (FA) genes, FANCA and FANCC, are hypersensitive to inflammatory cytokines. One of these, tumor necrosis factor α (TNF-α), is also overproduced by FA mononuclear phagocytes in response to certain Toll-like receptor (TLR) agonists, creating an autoinhibitory loop that may contribute to the pathogenesis of progressive bone marrow (BM) failure and selection of TNF-α-resistant leukemic stem cell clones. In macrophages, the TNF-α overproduction phenotype depends on p38 mitogen-activated protein kinase (MAPK), an enzyme also known to induce expression of other inflammatory cytokines, including interleukin 1β (IL-1β). Reasoning that IL-1β might be involved in a like autoinhibitory loop, we determined that (1) TLR activation of FANCA- and FANCC-deficient macrophages induced overproduction of both TNF-α and IL-1β in a p38-dependent manner; (2) exposure of Fancc-deficient BM progenitors to IL-1β potently suppressed the expansion of multipotent progenitor cells in vitro; and (3) although TNF-α overexpression in FA cells is controlled posttranscriptionally by the p38 substrate MAPKAPK-2, p38-dependent overproduction of IL-1β is controlled transcriptionally. We suggest that multiple inflammatory cytokines overproduced by FANCA- and FANCC-deficient mononuclear phagocytes may contribute to the progressive BM failure that characterizes FA, and that to achieve suppression of this proinflammatory state, p38 is a more promising molecular therapeutic target than either IL-1β or TNF-α alone.

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

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Morris, Dale L; O'Neil, Shawn P; Devraj, Rajesh V; Portanova, Joseph P; Gilles, Richard W; Gross, Cindy J; Curtiss, Sandra W; Komocsar, Wendy J; Garner, Debra S; Happa, Fernando A; Kraus, Lori J; Nikula, Kristen J; Monahan, Joseph B; Selness, Shaun R; Galluppi, Gerald R; Shevlin, Kimberly M; Kramer, Jeffrey A; Walker, John K; Messing, Dean M; Anderson, David R; Mourey, Robert J; Whiteley, Laurence O; Daniels, John S; Yang, Jerry Z; Rowlands, Philip C; Alden, Carl L; Davis, John W; Sagartz, John E

2010-06-01

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Crosstalk between p38, Hsp25 and Akt in spinal motor neurons after sciatic nerve injury

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Murashov, A. K.; Ul Haq, I.; Hill, C.; Park, E.; Smith, M.; Wang, X.; Wang, X.; Goldberg, D. J.; Wolgemuth, D. J.

2001-01-01

The p38 stress-activated protein kinase pathway is involved in regulation of phosphorylation of Hsp25, which in turn regulates actin filament dynamic in non-neuronal cells. We report that p38, Hsp25 and Akt signaling pathways were specifically activated in spinal motor neurons after sciatic nerve axotomy. The activation of the p38 kinase was required for induction of Hsp25 expression. Furthermore, Hsp25 formed a complex with Akt, a member of PI-3 kinase pathway that prevents neuronal cell death. Together, our observations implicate Hsp25 as a central player in a complex system of signaling that may both promote regeneration of nerve fibers and prevent neuronal cell death in the injured spinal cord.

Analysis of Kinase Gene Expression in the Frontal Cortex of Suicide Victims: Implications of Fear and Stress

Directory of Open Access Journals (Sweden)

Kwang eChoi

2011-07-01

Full Text Available Suicide is a serious public health issue that results from an interaction between multiple risk factors including individual vulnerabilities to complex feelings of hopelessness, fear and stress. Although kinase genes have been implicated in fear and stress, including the consolidation and extinction of fearful memories, expression profiles of those genes in the brain of suicide victims are less clear. Using gene expression microarray data from the Online Stanley Genomics Database (www.stanleygenomics.org and a quantitative PCR, we investigated the expression profiles of multiple kinase genes including the calcium calmodulin-dependent kinase (CAMK, the cyclin-dependent kinase (CDK, the mitogen-activated protein kinase (MAPK, and the protein kinase C (PKC in the prefrontal cortex (PFC of mood disorder patients died with suicide (n=45 and without suicide (N=38. We also investigated the expression pattern of the same genes in the PFC of developing humans ranging in age from birth to 49 year (n=46. The expression levels of CAMK2B, CDK5, MAPK9, and PRKCI were increased in the PFC of suicide victims as compared to non-suicide controls (FDR-adjusted p < 0.05, fold change > 1.1. Those genes also showed changes in expression pattern during the postnatal development (FDR-adjusted p < 0.05. These results suggest that multiple kinase genes undergo age-dependent changes in normal brains as well as pathological changes in suicide brains. These findings may provide an important link to protein kinases known to be important for the development of fear memory, stress-associated neural plasticity and up-regulation in the PFC of suicide victims. More research is needed to better understand the functional role of these kinase genes that may be associated with the pathophysiology of suicide.

The Mitogen-Activated Protein Kinase p38 alpha Regulates Tubular Damage in Murine Anti-Glomerular Basement Membrane Nephritis

NARCIS (Netherlands)

Mueller, Ralf; Daniel, Christoph; Hugo, Christian; Amann, Kerstin; Mielenz, Dirk; Endlich, Karlhans; Braun, Tobias; van der Veen, Betty; Heeringa, Peter; Schett, Georg; Zwerina, Jochen

2013-01-01

p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus

Epithelial control of gut-associated lymphoid tissue formation through p38α-dependent restraint of NF-κB signaling

Science.gov (United States)

Caballero-Franco, Celia; Guma, Monica; Choo, Min-Kyung; Sano, Yasuyo; Enzler, Thomas; Karin, Michael; Mizoguchi, Atsushi; Park, Jin Mo

2015-01-01

The protein kinase p38α mediates cellular responses to environmental and endogenous cues that direct tissue homeostasis and immune responses. Studies of mice lacking p38α in several different cell types have demonstrated that p38α signaling is essential to maintaining the proliferation-differentiation balance in developing and steady-state tissues. The mechanisms underlying these roles involve cell-autonomous control of signaling and gene expression by p38α. Here we show that p38α regulates gut-associated lymphoid tissue (GALT) formation in a non-cell-autonomous manner. From an investigation of mice with intestinal epithelial cell-specific deletion of the p38α gene, we find that p38α serves to limit NF-κB signaling and thereby attenuate GALT-promoting chemokine expression in the intestinal epithelium. Loss of this regulation results in GALT hyperplasia and, in some animals, mucosa-associated B cell lymphoma. These anomalies occur independently of luminal microbial stimuli and are likely driven by direct epithelial-lymphoid interactions. Our study illustrates a novel p38α-dependent mechanism preventing excessive generation of epithelial-derived signals that drive lymphoid tissue overgrowth and malignancy. PMID:26792803

FANCA and FANCC modulate TLR and p38 MAPK–dependent expression of IL-1β in macrophages

Science.gov (United States)

Garbati, Michael R.; Hays, Laura E.; Keeble, Winifred; Yates, Jane E.; Rathbun, R. Keaney

2013-01-01

Hematopoietic stem and progenitor cells with inactivated Fanconi anemia (FA) genes, FANCA and FANCC, are hypersensitive to inflammatory cytokines. One of these, tumor necrosis factor α (TNF-α), is also overproduced by FA mononuclear phagocytes in response to certain Toll-like receptor (TLR) agonists, creating an autoinhibitory loop that may contribute to the pathogenesis of progressive bone marrow (BM) failure and selection of TNF-α–resistant leukemic stem cell clones. In macrophages, the TNF-α overproduction phenotype depends on p38 mitogen-activated protein kinase (MAPK), an enzyme also known to induce expression of other inflammatory cytokines, including interleukin 1β (IL-1β). Reasoning that IL-1β might be involved in a like autoinhibitory loop, we determined that (1) TLR activation of FANCA- and FANCC-deficient macrophages induced overproduction of both TNF-α and IL-1β in a p38-dependent manner; (2) exposure of Fancc-deficient BM progenitors to IL-1β potently suppressed the expansion of multipotent progenitor cells in vitro; and (3) although TNF-α overexpression in FA cells is controlled posttranscriptionally by the p38 substrate MAPKAPK-2, p38-dependent overproduction of IL-1β is controlled transcriptionally. We suggest that multiple inflammatory cytokines overproduced by FANCA- and FANCC-deficient mononuclear phagocytes may contribute to the progressive BM failure that characterizes FA, and that to achieve suppression of this proinflammatory state, p38 is a more promising molecular therapeutic target than either IL-1β or TNF-α alone. PMID:24046015

TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture.

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Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-04-30

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFβ1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFβ1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFβ1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFβ1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFβ1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFβ1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFβ1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFβ1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFβ1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative

TRAF6 promotes myogenic differentiation via the TAK1/p38 mitogen-activated protein kinase and Akt pathways.

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Fang Xiao

Full Text Available p38 mitogen-activated protein kinase (MAPK is an essential kinase involved in myogenic differentiation. Although many substrates of p38 MAPK have been identified, little is known about its upstream activators during myogenic differentiation. TRAF6 is known to function in cytokine signaling during inflammatory responses. However, not much is known about its role in myogenic differentiation and muscle regeneration. We showed here that TRAF6 and its intrinsic ubiquitin E3 ligase activity are required for myogenic differentiation. In mouse myoblasts, knockdown of TRAF6 compromised the p38 MAPK and Akt pathways, while deliberate activation of either pathway rescued the differentiation defect caused by TRAF6 knockdown. TAK1 acted as a key signal transducer downstream of TRAF6 in myogenic differentiation. In vivo, knockdown of TRAF6 in mouse muscles compromised the injury-induced muscle regeneration without impairing macrophage infiltration and myoblast proliferation. Collectively, we demonstrated that TRAF6 promotes myogenic differentiation and muscle regeneration via the TAK1/p38 MAPK and Akt pathways.

Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

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Calviño, Eva; Estañ, María Cristina; Sánchez-Martín, Carlos; Brea, Rocío; de Blas, Elena; Boyano-Adánez, María del Carmen; Rial, Eduardo; Aller, Patricio

2014-02-01

3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 μM and a pure necrotic response at 60 μM. Low concentrations of 3-BrP (10-20 μM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 μM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy.

Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases.

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Yoshizumi, Masanori; Abe, Jun-Ichi; Tsuchiya, Koichiro; Berk, Bradford C; Tamaki, Toshiaki

2003-03-01

Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-alpha regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm(2)) on TNF-alpha-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-alpha activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-alpha activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-alpha. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

Epithelial Control of Gut-Associated Lymphoid Tissue Formation through p38α-Dependent Restraint of NF-κB Signaling.

Science.gov (United States)

Caballero-Franco, Celia; Guma, Monica; Choo, Min-Kyung; Sano, Yasuyo; Enzler, Thomas; Karin, Michael; Mizoguchi, Atsushi; Park, Jin Mo

2016-03-01

The protein kinase p38α mediates cellular responses to environmental and endogenous cues that direct tissue homeostasis and immune responses. Studies of mice lacking p38α in several different cell types have demonstrated that p38α signaling is essential to maintaining the proliferation-differentiation balance in developing and steady-state tissues. The mechanisms underlying these roles involve cell-autonomous control of signaling and gene expression by p38α. In this study, we show that p38α regulates gut-associated lymphoid tissue (GALT) formation in a noncell-autonomous manner. From an investigation of mice with intestinal epithelial cell-specific deletion of the p38α gene, we find that p38α serves to limit NF-κB signaling and thereby attenuate GALT-promoting chemokine expression in the intestinal epithelium. Loss of this regulation results in GALT hyperplasia and, in some animals, mucosa-associated B cell lymphoma. These anomalies occur independently of luminal microbial stimuli and are most likely driven by direct epithelial-lymphoid interactions. Our study illustrates a novel p38α-dependent mechanism preventing excessive generation of epithelial-derived signals that drive lymphoid tissue overgrowth and malignancy. Copyright © 2016 by The American Association of Immunologists, Inc.

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

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

2017-06-01

Obesity often leads to obesity-related cardiac hypertrophy (ORCH), which is suppressed by zinc-induced inactivation of p38 mitogen-activated protein kinase (p38 MAPK). In this study, we investigated the mechanisms by which zinc inactivates p38 MAPK to prevent ORCH. Mice (4-week old) were fed either high fat diet (HFD, 60% kcal fat) or normal diet (ND, 10% kcal fat) containing variable amounts of zinc (deficiency, normal and supplement) for 3 and 6 months. P38 MAPK siRNA and the p38 MAPK inhibitor SB203580 were used to suppress p38 MAPK activity in vitro and in vivo, respectively. HFD activated p38 MAPK and increased expression of B-cell lymphoma/CLL 10 (BCL10) and caspase recruitment domain family member 9 (CARD9). These responses were enhanced by zinc deficiency and attenuated by zinc supplement. Administration of SB203580 to HFD mice or specific siRNA in palmitate-treated cardiomyocytes eliminated the HFD and zinc deficiency activation of p38 MAPK, but did not significantly impact the expression of BCL10 and CARD9. In cultured cardiomyocytes, inhibition of BCL10 expression by siRNA prevented palmitate-induced increased p38 MAPK activation and atrial natriuretic peptide (ANP) expression. In contrast, inhibition of p38 MAPK prevented ANP expression, but did not affect BCL10 expression. Deletion of metallothionein abolished the protective effect of zinc on palmitate-induced up-regulation of BCL10 and phospho-p38 MAPK. HFD and zinc deficiency synergistically induce ORCH by increasing oxidative stress-mediated activation of BCL10/CARD9/p38 MAPK signalling. Zinc supplement ameliorates ORCH through activation of metallothionein to repress oxidative stress-activated BCL10 expression and p38 MAPK activation. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Novel p38α MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule library

DEFF Research Database (Denmark)

Petersen, L. K.; Blakskjær, P.; Chaikuad, A.

2016-01-01

A highly specific and potent (7 nM cellular IC50) inhibitor of p38α kinase was identified directly from a 12.6 million membered DNA-encoded small molecule library. This was achieved using the high fidelity yoctoReactor technology (yR) for preparing the DNA-encoded library, and a homogeneous...... interactions. Moreover, the crystal structure showed, that although buried in the p38α active site, the original DNA attachment point of the compound was accessible through a channel created by the distorted P-loop conformation. This study demonstrates the usability of DNA-encoded library technologies...

Carprofen Induction of p75NTR Dependent Apoptosis via the p38 MAPK Pathway in Prostate Cancer Cells

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Khwaja, Fatima S.; Quann, Emily J.; Pattabiraman, Nagarajan; Wynne, Shehla; Djakiew, Daniel

2008-01-01

The p75NTR functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we demonstrated that treatment with R-flurbiprofen or ibuprofen induced p75NTR expression in several prostate cancer cell lines leading to p75NTR mediated decreased survival. Utilizing the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico data base of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75NTR levels and inhibition of cell survival. Prostate (PC-3, DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75NTR associated loss of survival than breast (MCF7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant negative form of p75NTR prior to carprofen treatment partially rescued cell survival demonstrating a cause and effect relationship between carprofen induction of p75NTR levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF7 and 3T3 cells. Furthermore, siRNA knockdown of the p38 MAPK protein prevented induction of p75NTR by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 minute. Expression of a dominant negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75NTR protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75NTR dependent apoptosis via the p38 MAPK pathway in prostate cancer cells. PMID:18974393

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans*

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Andrusiak, Matthew G.; Jin, Yishi

2016-01-01

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundworm Caenorhabditis elegans was developed as a system to study genes required for development and nervous system function. The powerful genetics of C. elegans in combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components in C. elegans. PMID:26907690

Puerarin reduces apoptosis in rat hippocampal neurons culturea in high glucose medium by modulating the p38 mitogen activated protein kinase and c-Jun N-terminal kinase signaling pathways.

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Xu, Xiaohan; Wang, Jingbo; Zhang, Hong; Tian, Guoqing; Liu, Yuqin

2016-02-01

To investigate the neuroprotective etfect of puerarin on rat hippocampal neurons cultured in high glucose medium, and to examine the role of the p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways in this effect. Primary cultures of hippocampal neurons were prepared from newborn Sprague Dawley rats. Neuron-specific enolase immunocytochemistry was used to identify neurons. The neurons were cultured with normal medium (control group) or with high-glucose medium (high-glucose group), and puerarin (puerarin group), a p38 MAPK inhibitor (SB239063; p38 MAPK inhibitor group) or a JNK inhibitor (SP600125; JNK inhibitor group) were added. After 72 h of treatment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to detect apoptosis, and western blotting was used to assess protein levels of p-p38, p38, p-JNK and JNK. In the high-glucose group, the neuronal apoptosis rate and the p-p38/p38 and p-JNK/JNK ratios were higher than in the control group. The p38 MAPK and JNK inhibitors prevented this increase in the apoptosis rate. The apoptosis rates in the puerarin group, the p38 MAPK inhibitor group and the JNK inhibitor group were significantly decreased compared with the high-glucose group. Moreover, protein levels of p-p38 and p-JNK were significantly reduced, and the p-p38/p38 and p-JNK/JNK ratios were decreased in the puerarin group compared with the high-glucose group. In addition, compared with the high-glucose group, p-p38 levels and the p-p38/p38 ratio were reduced in the p38 MAPK inhibitor group, and p-JNK levels and the p-JNK/JNK ratio were decreased in the JNK inhibitor group. Puerarin attenuates neuronal apoptosis induced by high glucose by reducing the phosphorylation of p38 and JNK.

On-line electrochemistry-bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38α kinase inhibitors.

Science.gov (United States)

Falck, David; de Vlieger, Jon S B; Giera, Martin; Honing, Maarten; Irth, Hubertus; Niessen, Wilfried M A; Kool, Jeroen

2012-04-01

In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a tracer (SKF-86002) that shows fluorescence enhancement in the orthosteric binding site of the p38α kinase is the readout for bioaffinity. Parallel HR-MS(n) experiments provided information on the identity of binders and non-binders. Finally, the data produced with this on-line system were compared to electrochemical conversion products generated off-line. The electrochemical conversion of 1-{6-chloro-5-[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazine-1-carbonyl]-3aH-indol-3-yl}-2-morpholinoethane-1,2-dione resulted in eight products, three of which showed bioaffinity in the continuous-flow p38α bioaffinity assay used. Electrochemical conversion of BIRB796 resulted, amongst others, in the formation of the reactive quinoneimine structure and its corresponding hydroquinone. Both products were detected in the p38α bioaffinity assay, which indicates binding to the p38α kinase.

Neuroprotective effects of Arctium lappa L. roots against glutamate-induced oxidative stress by inhibiting phosphorylation of p38, JNK and ERK 1/2 MAPKs in PC12 cells.

Science.gov (United States)

Tian, Xing; Sui, Shuang; Huang, Jin; Bai, Jun-Peng; Ren, Tian-Shu; Zhao, Qing-Chun

2014-07-01

Many studies have shown that glutamate-induced oxidative stress can lead to neuronal cell death involved in the development of neurodegenerative diseases. In this work, protective effects of ethyl acetate extract (EAE) of Arctium lappa L. roots against glutamate-induced oxidative stress in PC12 cells were evaluated. Also, the effects of EAE on antioxidant system, mitochondrial pathway, and signal transduction pathway were explored. Pretreatment with EAE significantly increased cell viability, activities of GSH-Px and SOD, mitochondrial membrane potential and reduced LDH leakage, ROS formation, and nuclear condensation in a dose-dependent manner. Furthermore, western blot results revealed that EAE increased the Bcl-2/Bax ratio, and inhibited the up-regulation of caspase-3, release of cytochrome c, phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK 1/2). Therefore, our results indicate that EAE may be a promising neuroprotective agent for the prevention and treatment of neurodegenerative diseases implicated with oxidative stress. Copyright © 2014 Elsevier B.V. All rights reserved.

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

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Andrusiak, Matthew G; Jin, Yishi

2016-04-08

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)2] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38MAPK/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment.

Science.gov (United States)

Filomeni, Giuseppe; Cardaci, Simone; Da Costa Ferreira, Ana Maria; Rotilio, Giuseppe; Ciriolo, Maria Rosa

2011-08-01

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N']copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment. © The Authors Journal compilation © 2011 Biochemical Society

p38 mitogen-activated protein kinase up-regulates NF-κB transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

International Nuclear Information System (INIS)

Ji, Guoping; Liu, Dongxu; Liu, Jing; Gao, Hui; Yuan, Xiao; Shen, Gang

2010-01-01

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-κB activation during myogenesis, not through down-regulation of degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that stretch-induced NF-κB activation by phosphorylation of p65 NF-κB. Moreover, depletion of p38α using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-κB activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-κB signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The α isoform of p38MAP kinase regulates the transcriptional activation of NF-κB following stimulation with cyclic stretch.

p38 mitogen-activated protein kinase up-regulates NF-{kappa}B transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

Energy Technology Data Exchange (ETDEWEB)

Ji, Guoping [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China); Liu, Dongxu [Department of Orthodontics, College of Stomatology, Shandong University, Jinan, Shandong Province 250012 (China); Liu, Jing [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Gao, Hui [Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041 (China); Yuan, Xiao, E-mail: yuanxiaoqd@163.com [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Shen, Gang, E-mail: ganshen2007@163.com [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China)

2010-01-01

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-{kappa}B in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-{kappa}B activation during myogenesis, not through down-regulation of degradation of I{kappa}B-{alpha}, and consequent translocation of the p65 subunit of NF-{kappa}B to the nucleus. It is likely that stretch-induced NF-{kappa}B activation by phosphorylation of p65 NF-{kappa}B. Moreover, depletion of p38{alpha} using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-{kappa}B activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-{kappa}B signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The {alpha} isoform of p38MAP kinase regulates the transcriptional activation of NF-{kappa}B following stimulation with cyclic stretch.

Synthesis of the highly selective p38 MAPK inhibitor UR-13756 for possible therapeutic use in Werner syndrome.

Science.gov (United States)

Bagley, Mark C; Davis, Terence; Rokicki, Michal J; Widdowson, Caroline S; Kipling, David

2010-02-01

UR-13756 is a potent and selective p38 mitogen-activated protein kinase (MAPK) inhibitor, reported to have good bioavailability and pharmacokinetic properties and, thus, is of potential use in the treatment of accelerated aging in Werner syndrome. Irradiation of 2-chloroacrylonitrile and methylhydrazine in ethanol at 100 °C gives 1-methyl-3-aminopyrazole, which reacts with 4-fluorobenzaldehyde and a ketone, obtained by Claisen condensation of 4-picoline, in a Hantzsch-type 3-component hereocyclocondensation, to give the pyrazolopyridine UR-13756. UR-13756 shows p38 MAPK inhibitory activity in human telomerase reverse transcriptase-immortalized HCA2 dermal fibroblasts, with an IC(50) of 80 nm, as shown by ELISA, is 100% efficacious for up to 24 h at 1.0 μm and displays excellent kinase selectivity over the related stress-activated c-Jun kinases. In addition, UR-13756 is an effective p38 inhibitor at 1.0 μm in Werner syndrome cells, as shown by immunoblot. The convergent synthesis of UR-13756 is realized using microwave dielectric heating and provides a highly selective inhibitor that shows excellent selectivity for p38 MAPK over c-Jun N-terminal kinase.

Hepatic p38α regulates gluconeogenesis by suppressing AMPK.

Science.gov (United States)

Jing, Yanyan; Liu, Wei; Cao, Hongchao; Zhang, Duo; Yao, Xuan; Zhang, Shengjie; Xia, Hongfeng; Li, Dan; Wang, Yu-cheng; Yan, Jun; Hui, Lijian; Ying, Hao

2015-06-01

It is proposed that p38 is involved in gluconeogenesis, however, the genetic evidence is lacking and precise mechanisms remain poorly understood. We sought to delineate the role of hepatic p38α in gluconeogenesis during fasting by applying a loss-of-function genetic approach. We examined fasting glucose levels, performed pyruvate tolerance test, imaged G6Pase promoter activity, as well as determined the expression of gluconeogenic genes in mice with a targeted deletion of p38α in liver. Results were confirmed both in vivo and in vitro by using an adenoviral dominant-negative form of p38α (p38α-AF) and the constitutively active mitogen-activated protein kinase 6, respectively. Adenoviral dominant-negative form of AMP-activated protein kinase α (DN-AMPKα) was employed to test our proposed model. Mice lacking hepatic p38α exhibited reduced fasting glucose level and impaired gluconeogenesis. Interestingly, hepatic deficiency of p38α did not result in an alteration in CREB phosphorylation, but led to an increase in AMPKα phosphorylation. Adenoviral DN-AMPKα could abolish the effect of p38α-AF on gluconeogenesis. Knockdown of up-steam transforming growth factor β-activated kinase 1 decreased the AMPKα phosphorylation induced by p38α-AF, suggesting a negative feedback loop. Consistently, inverse correlations between p38 and AMPKα phosphorylation were observed during fasting and in diabetic mouse models. Importantly, adenoviral p38α-AF treatment ameliorated hyperglycemia in diabetic mice. Our study provides evidence that hepatic p38α functions as a negative regulator of AMPK signaling in maintaining gluconeogenesis, dysregulation of this regulatory network contributes to unrestrained gluconeogenesis in diabetes, and hepatic p38α could be a drug target for hyperglycemia. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Oxidative stress by layered double hydroxide nanoparticles via an SFK-JNK and p38-NF-κB signaling pathway mediates induction of interleukin-6 and interleukin-8 in human lung epithelial cells

Directory of Open Access Journals (Sweden)

Choi SJ

2015-04-01

Full Text Available Soo-Jin Choi, Hee-Jeong Paek, Jin YuDepartment of Food Science and Technology, Seoul Women’s University, Seoul, Republic of KoreaAbstract: Anionic nanoclays are layered double hydroxide nanoparticles (LDH-NPs that have been shown to exhibit toxicity by inducing reactive oxidative species and a proinflammatory mediator in human lung epithelial A549 cells. However, the molecular mechanism responsible for this LDH-NP-induced toxicity and the relationship between oxidative stress and inflammatory events remains unclear. In this study, we focused on intracellular signaling pathways and transcription factors induced in response to oxidative stress caused by exposure to LDH-NPs in A549 cells. Mitogen-activated protein kinase (MAPK cascades, such as extracellular signal-regulated kinase, c-Jun-N-terminal kinase (JNK, and p38, were investigated as potential signaling mechanisms responsible for regulation of oxidative stress and cytokine release. Src family kinases (SFKs, which are known to mediate activation of MAPK, together with redox-sensitive transcription factors, including nuclear factor kappa B and nuclear factor-erythroid 2-related factor-2, were also investigated as downstream events of MAPK signaling. The results obtained suggest that LDH-NP exposure causes oxidative stress, leading to expression of antioxidant enzymes, such as catalase, glucose reductase, superoxide dismutase, and heme oxygenase-1, via a SFK-JNK and p38-nuclear factor kappa B signaling pathway. Further, activation of this signaling was also found to regulate release of inflammatory cytokines, including interleukin-6 and interleukin-8, demonstrating the inflammatory potential of LDH-NP.Keywords: layered double hydroxide, mitogen-activated protein kinases, Src family kinases, nuclear factor kappa B, oxidative stress, inflammatory cytokine

Lysyl Oxidase Induces Vascular Oxidative Stress and Contributes to Arterial Stiffness and Abnormal Elastin Structure in Hypertension: Role of p38MAPK.

Science.gov (United States)

Martínez-Revelles, Sonia; García-Redondo, Ana B; Avendaño, María S; Varona, Saray; Palao, Teresa; Orriols, Mar; Roque, Fernanda R; Fortuño, Ana; Touyz, Rhian M; Martínez-González, Jose; Salaices, Mercedes; Rodríguez, Cristina; Briones, Ana M

2017-09-01

Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H 2 O 2 as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with β-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H 2 O 2 and O 2 .- levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mito-TEMPO, the antioxidant apocynin, or the H 2 O 2 scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mito-TEMPO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. Antioxid. Redox Signal. 27

P38 mitogen-activated protein kinase (p38 MAPK) overexpression in clinical staging of nasopharyngeal carcinoma

Science.gov (United States)

Farhat; Asnir, R. A.; Yudhistira, A.; Daulay, E. R.; Muzakkir, M. M.; Yulius, S.

2018-03-01

Molecular biological research on nasopharyngeal carcinoma has been widely practiced, such as VEGF, EGFR, COX-2 expression and so on. MAPK plays a role in cell growth such as proliferation, differentiation, and apoptosis, primarily contributing to gene expression, where p38 MAPK pathway mostly associate with anti-apoptosis and cause cell transformation. The aim of this study is to determine the expression of p38 MAPK in clinical stage of nasopharyngeal carcinoma so that the result can be helpful in prognosis and adjunctive therapy in nasopharyngeal carcinoma. The research design is descriptive. It was done in THT- KL Department of FK USU/RSUP Haji Adam Malik, Medan and Pathology Anatomical Department of FK USU. The study was conducted from December 2011 to May 2012. The Samples are all patients who diagnosed with nasopharyngeal carcinoma in oncology division of Otorhinolaryngology Department. p38 MAPK overexpression was found in 21 samples (70%) from 30 nasopharyngeal carcinoma samples. The elevated of p38 MAPK expression most found on T4 by eight samples (38.1%), N3 lymph node group by nine samples (42.9%), stage IV of clinical staging is as many as 15 samples (71.4%). p38 MAPK most expressed in stage IV clinical staging of patients with nasopharyngeal carcinoma.

Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Science.gov (United States)

Lin, Chih-Chung; Lee, I-Ta; Hsu, Chun-Hao; Hsu, Chih-Kai; Chi, Pei-Ling; Hsiao, Li-Der; Yang, Chuen-Mao

2015-01-01

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation. PMID:25734900

EphA2 modulates radiosensitive of hepatocellular carcinoma cells via p38/mitogen-activated protein kinase-mediated signal pathways

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Qiao Jin

2015-10-01

Full Text Available This experiment was conducted to investigate the role of EPH receptor A2 (EphA2 in the modulation of radiosensitivity of hepatic cellular cancer (HCC cells and to determine whether p38/mitogen-activated protein kinase (p38MAPK signaling mediated EphA2 function in this respect. The protein expressions of EphA2 and phosphorylated p38MAPK were tested in HCC and normal hepatic tissues. In HCC 97H cells, EphA2 was overexpressed and knocked out by transfection with EphA2 expression vector and EphA2-ShRNA, respectively, prior to cell exposure to low-dose irradiation. Significantly upregulated EphA2 and phosphorylated p38MAPK were observed in HCC tissues, compared with those in normal hepatic tissues. Low-dose irradiation (1 Gy only caused minor damage to HCC 97H cells, as assessed by alterations in cell viability, apoptosis rate, and cell healing capacity (p = 0.072, p = 0.078, and p = 0.069 respectively. However, EphA2 knock-out in HCC 97H cells induced significant reduction in cell viability and cell healing capacity after these cells were subjected to low-dose irradiation. Apoptosis rate underwent dramatic increase (p < 0.01. By contrast, EphA2 overexpression in HCC 97H cells reversed these effects and enhanced cell colony formation rate, thus displaying remarkable attenuation of radiosensitivity of HCC 97H cells. Further, SB203580, a specific inhibitor of p38MAPK, was added to HCC 97H cells over-expressing EphA2. The effect of EphA2 overexpression on the radiosensitivity of HCC 97H cells was abrogated. Thus, the present study indicates that EphA2 have the ability to negatively regulate the radiosensitivity of HCC 97H cells, which mainly depends on 38MAPK-mediated signal pathways.

Aged dominant negative p38α MAPK mice are resistant to age-dependent decline in adult-neurogenesis and context discrimination fear conditioning.

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Cortez, IbDanelo; Bulavin, Dmitry V; Wu, Ping; McGrath, Erica L; Cunningham, Kathryn A; Wakamiya, Maki; Papaconstantinou, John; Dineley, Kelly T

2017-03-30

A major aspect of mammalian aging is the decline in functional competence of many self-renewing cell types, including adult-born neuronal precursors. Since age-related senescence of self-renewal occurs simultaneously with chronic up-regulation of the p38MAPKalpha (p38α) signaling pathway, we used the dominant negative mouse model for attenuated p38α activity (DN-p38α AF/+ ) in which Thr180 and Tyr182 are mutated (T→A/Y→F) to prevent phosphorylation activation (DN-p38α AF/+ ) and kinase activity. As a result, aged DN-p38α AF/+ mice are resistant to age-dependent decline in proliferation and regeneration of several peripheral tissue progenitors when compared to wild-type littermates. Aging is the major risk factor for non-inherited forms of Alzheimer's disease (AD); environmental and genetic risk factors that accelerate the senescence phenotype are thought to contribute to an individual's relative risk. In the present study, we evaluated aged DN-p38α AF/+ and wildtype littermates in a series of behavioral paradigms to test if p38α mutant mice exhibit altered baseline abnormalities in neurological reflexes, locomotion, anxiety-like behavior, and age-dependent cognitive decline. While aged DN-p38α AF/+ and wildtype littermates appear equal in all tested baseline neurological and behavioral parameters, DN-p38α AF/+ exhibit superior context discrimination fear conditioning. Context discrimination is a cognitive task that is supported by proliferation and differentiation of adult-born neurons in the dentate gyrus of the hippocampus. Consistent with enhanced context discrimination in aged DN-p38α AF/+ , we discovered enhanced production of adult-born neurons in the dentate gyrus of DN-p38α AF/+ mice compared to wildtype littermates. Our findings support the notion that p38α inhibition has therapeutic utility in aging diseases that affect cognition, such as AD. Copyright © 2016 Elsevier B.V. All rights reserved.

Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

Science.gov (United States)

Sung, Ho Joong; Son, Sin Jee; Yang, Seung-ju; Rhee, Ki-Jong; Kim, Yoon Suk

2012-07-01

Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.

Regulation of p73 by Hck through kinase-dependent and independent mechanisms

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Radha Vegesna

2007-05-01

Full Text Available Abstract Background p73, a p53 family member is a transcription factor that plays a role in cell cycle, differentiation and apoptosis. p73 is regulated through post translational modifications and protein interactions. c-Abl is the only known tyrosine kinase that phosphorylates and activates p73. Here we have analyzed the role of Src family kinases, which are involved in diverse signaling pathways, in regulating p73. Results Exogenously expressed as well as cellular Hck and p73 interact in vivo. In vitro binding assays show that SH3 domain of Hck interacts with p73. Co-expression of p73 with Hck or c-Src in mammalian cells resulted in tyrosine phosphorylation of p73. Using site directed mutational analysis, we determined that Tyr-28 was the major site of phosphorylation by Hck and c-Src, unlike c-Abl which phosphorylates Tyr-99. In a kinase dependent manner, Hck co-expression resulted in stabilization of p73 protein in the cytoplasm. Activation of Hck in HL-60 cells resulted in tyrosine phosphorylation of endogenous p73. Both exogenous and endogenous Hck localize to the nuclear as well as cytoplasmic compartment, just as does p73. Ectopically expressed Hck repressed the transcriptional activity of p73 as determined by promoter assays and semi-quantitative RT-PCR analysis of the p73 target, Ipaf and MDM2. SH3 domain- dependent function of Hck was required for its effect on p73 activity, which was also reflected in its ability to inhibit p73-mediated apoptosis. We also show that Hck interacts with Yes associated protein (YAP, a transcriptional co-activator of p73, and shRNA mediated knockdown of YAP protein reduces p73 induced Ipaf promoter activation. Conclusion We have identified p73 as a novel substrate and interacting partner of Hck and show that it regulates p73 through mechanisms that are dependent on either catalytic activity or protein interaction domains. Hck-SH3 domain-mediated interactions play an important role in the inhibition of p73

Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium

DEFF Research Database (Denmark)

Marshall, W. S.; Ossum, Carlo Gunnar; Hoffmann, Else Kay

2005-01-01

analysis) by eightfold at 5 min, then more slowly again to sevenfold at 60 min. Hypertonic shock slowly increased p38 by sevenfold at 60 min. Phosphorylated JNK kinase was increased by 40-50% by both hypotonic and hypertonic shock and was still elevated at 30 min in hypertonic medium. By immunoblot...... analysis it was found that the stress protein kinase (SPAK) and oxidation stress response kinase 1 (OSR1) were present in salt and freshwater acclimated fish with higher expression in freshwater. By immunocytochemistry, SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized with NKCC...

p38- and MK2-dependent signalling promotes stress-induced centriolar satellite remodelling via 14-3-3-dependent sequestration of CEP131/AZI1

DEFF Research Database (Denmark)

Tollenaere, Maxim A X; Villumsen, Bine H; Blasius, Melanie

2015-01-01

Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trig...

Neuronal phosphorylated RNA-dependent protein kinase in Creutzfeldt-Jakob disease.

LENUS (Irish Health Repository)

Paquet, Claire

2009-02-01

The mechanisms of neuronal apoptosis in Creutzfeldt-Jakob disease (CJD) and their relationship to accumulated prion protein (PrP) are unclear. A recent cell culture study showed that intracytoplasmic PrP may induce phosphorylated RNA-dependent protein kinase (PKR(p))-mediated cell stress. The double-stranded RNA protein kinase PKR is a proapoptotic and stress kinase that accumulates in degenerating neurons in Alzheimer disease. To determine whether neuronal apoptosis in human CJD is associated with activation of the PKR(p) signaling pathway, we assessed in situ end labeling and immunocytochemistry for PrP, glial fibrillary acidic protein, CD68, activated caspase 3, and phosphorylated PKR (Thr451) in samples of frontal, occipital, and temporal cortex, striatum, and cerebellum from 6 patients with sporadic CJD and 5 controls. Neuronal immunostaining for activated PKR was found in all CJD cases. The most staining was in nuclei and, in contrast to findings in Alzheimer disease, cytoplasmic labeling was not detected. Both the number and distribution of PKR(p)-positive neurons correlated closely with the extent of neuronal apoptosis, spongiosis, astrocytosis, and microglial activation and with the phenotype and disease severity. There was no correlation with the type, topography, or amount of extracellular PrP deposits. These findings suggest that neuronal apoptosis in human CJD may result from PKR(p)-mediated cell stress and are consistent with recent studies supporting a pathogenic role for intracellular or transmembrane PrP.

The MAP kinase-activated protein kinase Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen Candida albicans.

Science.gov (United States)

Li, Xichuan; Du, Wei; Zhao, Jingwen; Zhang, Lilin; Zhu, Zhiyan; Jiang, Linghuo

2010-06-01

Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

Data on the phosphorylation of p38MAPK and JNK induced by chlorpyrifos in Drosophila melanogaster

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J.E.S. Batista

2016-12-01

Full Text Available Exposure to organophosphate compounds, such as chlorpyrifos, has been linked to disturbances on cell signaling pathways. Mitogen activated protein kinases (MAPK are a family of protein kinases involved in a range of cellular processes, including stress response, apoptosis and survival. Therefore, changes in the activation state of these kinases may characterize key mechanisms of toxicity elicited by xenobiotics. Here we report data on the phosphorylation of p38MAPK and JNK, members of the MAPK family, in Drosophila melanogaster exposed to chlorpyrifos, as characterized by western blotting assays.

In Silico Screening and In Vitro Activity Measurement of Javamide Analogues as Potential p38 MAPK Inhibitors.

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Park, Jae B

2017-12-13

p38 Mitogen-activated protein kinase (p38 MAPK) is a protein kinase critically involved in the progress of inflammation/stress-associated diseases. Our data suggested that javamide analogues may contain strong anti-inflammation activities, but there is little information about their effects on p38 MAPK. Therefore, in this paper, the effects of thirty javamide analogues on p38 MAPK were investigated using in silico screening and in vitro p38 MAPK assay methods. The javamide analogues were synthesized and their chemical structures were confirmed using nuclear magnetic resonance (NMR) spectroscopic methods. Then, the javamide analogues were screened using an in silico modeling program. The screened analogues demonstrated a wide range of binding energy (ΔE; -20 to -39) and several analogues with ΔE; -34 to -39 showed strong binding affinity to p38 MAPK. In vitro p38 MAPK assay, the kinase was significantly inhibited by the analogues with great binding energy (ΔE; -34 to -39) and in silico scores (Avg. score; -27.5 to -29.3). Furthermore, the comparative analysis of both assays showed a positive correlation between the in silico scores and p38 MAPK inhibition. In fact, the javamide analogues with top five in silico scores (Avg. score; -27.5 to -29.3) were found to inhibit p38 MAPK by 27-31% ( p silico score (Avg. score; -29.2) inhibited p38 MAPK (IC 50 = 9.9 μM) a little better than its methyl ester with best in silico score (Avg. score; -29.3). To support the ability to inhibit p38 MAPK, the treatment of javamide-II-ethyl and -methyl esters could suppress the production of IL-8 and MCP-1 protein significantly by 22-73% ( p silico and in vitro assay approach may be a useful and efficient solution as a functional screening approach in searching new lead compounds for targeted molecules.

Mesothelioma Cells Escape Heat Stress by Upregulating Hsp40/Hsp70 Expression via Mitogen-Activated Protein Kinases

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Michael Roth

2009-01-01

Full Text Available Therapy with hyperthermal chemotherapy in pleural diffuse malignant mesothelioma had limited benefits for patients. Here we investigated the effect of heat stress on heat shock proteins (HSP, which rescue tumour cells from apoptosis. In human mesothelioma and mesothelial cells heat stress (39–42°C induced the phosphorylation of two mitogen activated kinases (MAPK Erk1/2 and p38, and increased Hsp40, and Hsp70 expression. Mesothelioma cells expressed more Hsp40 and were less sensitive to heat stress compared to mesothelial cells. Inhibition of Erk1/2 MAPK by PD98059 or by Erk1 siRNA down-regulated heat stress-induced Hsp40 and Hsp70 expression and reduced mesothelioma cell survival. Inhibition of p38MAPK by SB203580 or siRNA reduced Hsp40, but not Hsp70, expression and also increased mesothelioma cell death. Thus hyperthermia combined with suppression of p38 MAPK or Hsp40 may represent a novel approach to improve mesothelioma therapy.

Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases

DEFF Research Database (Denmark)

Kampen, G T; Stafford, S; Adachi, T

2000-01-01

Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3...... and the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to assess...... the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay. Chemotaxis...

Basolateral amygdala bidirectionally modulates stress-induced hippocampal learning and memory deficits through a p25/Cdk5-dependent pathway.

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Rei, Damien; Mason, Xenos; Seo, Jinsoo; Gräff, Johannes; Rudenko, Andrii; Wang, Jun; Rueda, Richard; Siegert, Sandra; Cho, Sukhee; Canter, Rebecca G; Mungenast, Alison E; Deisseroth, Karl; Tsai, Li-Huei

2015-06-09

Repeated stress has been suggested to underlie learning and memory deficits via the basolateral amygdala (BLA) and the hippocampus; however, the functional contribution of BLA inputs to the hippocampus and their molecular repercussions are not well understood. Here we show that repeated stress is accompanied by generation of the Cdk5 (cyclin-dependent kinase 5)-activator p25, up-regulation and phosphorylation of glucocorticoid receptors, increased HDAC2 expression, and reduced expression of memory-related genes in the hippocampus. A combination of optogenetic and pharmacosynthetic approaches shows that BLA activation is both necessary and sufficient for stress-associated molecular changes and memory impairments. Furthermore, we show that this effect relies on direct glutamatergic projections from the BLA to the dorsal hippocampus. Finally, we show that p25 generation is necessary for the stress-induced memory dysfunction. Taken together, our data provide a neural circuit model for stress-induced hippocampal memory deficits through BLA activity-dependent p25 generation.

Basolateral amygdala bidirectionally modulates stress-induced hippocampal learning and memory deficits through a p25/Cdk5-dependent pathway

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Rei, Damien; Mason, Xenos; Seo, Jinsoo; Gräff, Johannes; Rudenko, Andrii; Wang, Jun; Rueda, Richard; Siegert, Sandra; Cho, Sukhee; Canter, Rebecca G.; Mungenast, Alison E.; Deisseroth, Karl; Tsai, Li-Huei

2015-01-01

Repeated stress has been suggested to underlie learning and memory deficits via the basolateral amygdala (BLA) and the hippocampus; however, the functional contribution of BLA inputs to the hippocampus and their molecular repercussions are not well understood. Here we show that repeated stress is accompanied by generation of the Cdk5 (cyclin-dependent kinase 5)-activator p25, up-regulation and phosphorylation of glucocorticoid receptors, increased HDAC2 expression, and reduced expression of memory-related genes in the hippocampus. A combination of optogenetic and pharmacosynthetic approaches shows that BLA activation is both necessary and sufficient for stress-associated molecular changes and memory impairments. Furthermore, we show that this effect relies on direct glutamatergic projections from the BLA to the dorsal hippocampus. Finally, we show that p25 generation is necessary for the stress-induced memory dysfunction. Taken together, our data provide a neural circuit model for stress-induced hippocampal memory deficits through BLA activity-dependent p25 generation. PMID:25995364

Social interaction reward decreases p38 activation in the nucleus accumbens shell of rats.

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Salti, Ahmad; Kummer, Kai K; Sadangi, Chinmaya; Dechant, Georg; Saria, Alois; El Rawas, Rana

2015-12-01

We have previously shown that animals acquired robust conditioned place preference (CPP) to either social interaction alone or cocaine alone. Recently it has been reported that drugs of abuse abnormally activated p38, a member of mitogen-activated protein kinase family, in the nucleus accumbens. In this study, we aimed to investigate the expression of the activated form of p38 (pp38) in the nucleus accumbens shell and core of rats expressing either cocaine CPP or social interaction CPP 1 h, 2 h and 24 h after the CPP test. We hypothesized that cocaine CPP will increase pp38 in the nucleus accumbens shell/core as compared to social interaction CPP. Surprisingly, we found that 24 h after social interaction CPP, pp38 neuronal levels were decreased in the nucleus accumbens shell to the level of naïve rats. Control saline rats that received saline in both compartments of the CPP apparatus and cocaine CPP rats showed similar enhanced p38 activation as compared to naïve and social interaction CPP rats. We also found that the percentage of neurons expressing dopaminergic receptor D2R and pp38 was also decreased in the shell of the nucleus accumbens of social interaction CPP rats as compared to controls. Given the emerging role of p38 in stress/anxiety behaviors, these results suggest that (1) social interaction reward has anti-stress effects; (2) cocaine conditioning per se does not affect p38 activation and that (3) marginal stress is sufficient to induce p38 activation in the shell of the nucleus accumbens. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Efficacy and gastrointestinal tolerability of ML3403, a selective inhibitor of p38 MAP kinase and CBS-3595, a dual inhibitor of p38 MAP kinase and phosphodiesterase 4 in CFA-induced arthritis in rats.

Science.gov (United States)

Koch, Diana A; Silva, Rodrigo B M; de Souza, Alessandra H; Leite, Carlos E; Nicoletti, Natália F; Campos, Maria M; Laufer, Stefan; Morrone, Fernanda B

2014-03-01

Mitogen-activated protein kinase (MAPK) p38 inhibitors have entered the clinical phase, although many of them have failed due to high toxicity and lack of efficacy. In the present study we compared the effects of the selective p38 inhibitor ML3403 and the dual p38-PDE4 inhibitor CBS-3595, on inflammatory and nociceptive parameters in a model of polyarthritis in rats. Male Wistar rats (180-200 g) were used for the complete Freund's adjuvant (CFA)-induced arthritis model and they were evaluated at 14-21 days. We also analysed the effects of these pharmacological tools on liver and gastrointestinal toxicity and on cytokine levels. Repeated CBS-3595 (3 mg/kg) or ML3403 (10 mg/kg) administration produced significant anti-inflammatory actions in the chronic arthritis model induced by CFA. CBS-3595 and ML3403 treatment also markedly reduced the production of the proinflammatory cytokine IL-6 in the paw tissue, whereas it widely increased the levels of the anti-inflammatory cytokine IL-10. Moreover, CBS-3595 produced partial anti-allodynic effects in the CFA model at 4 and 8 days after treatment. Notably, ML3403 and CBS-3595 did not show marked signs of hepatoxicity, as supported by unaltered histological observations in the liver sections. Finally, both compounds were safe in the gastrointestinal tract, according to evaluation of intestinal biopsies. CBS-3595 displayed a superior profile regarding its anti-inflammatory effects. Thus p38 MAPK/PDE4 blocking might well constitute a relevant strategy for the treatment of RA.

Suppression of cadmium-induced JNK/p38 activation and HSP70 family gene expression by LL-Z1640-2 in NIH3T3 cells

International Nuclear Information System (INIS)

Sugisawa, Nobusuke; Matsuoka, Masato; Okuno, Takeo; Igisu, Hideki

2004-01-01

When NIH3T3 cells were exposed to CdCl 2 , the three major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase (ERK), c-Jun NH 2 -terminal kinase (JNK), and p38, were phosphorylated in a time (1-9 h)- and dose (1-20 μM)-dependent manner. Treatment with a macrocyclic nonaketide compound, LL-Z1640-2 (10-100 ng/ml), suppressed the phosphorylation of MAPKs without affecting the total protein level in cells exposed to 10 μM CdCl 2 for 6 h. CdCl 2 -induced phosphorylation of c-Jun on Ser63 and that on Ser73, and resultant accumulation of total c-Jun protein were also suppressed by LL-Z1640-2 treatment. The in vitro kinase assays also showed significant inhibitory effects of LL-Z1640-2 (at 10 or 25 ng/ml) on JNK and p38 but less markedly. In contrast to JNK and p38, ERK activity was inhibited moderately only at 50 or 100 ng/ml LL-Z1640-2. On the other hand, other JNK inhibitors, SP600125 and L-JNKI1, failed to suppress CdCl 2 -induced activation of the JNK pathway. Among the mouse stress response genes upregulated in response to CdCl 2 exposure, the expressions of hsp68 (encoding for heat shock 70 kDa protein 1; Hsp70-1) and grp78 (encoding for 78 kDa glucose-regulated protein; Grp78) genes were suppressed by treatment with 25 ng/ml LL-Z1640-2. Thus, LL-Z1640-2 could suppress CdCl 2 -induced activation of JNK/p38 pathways and expression of HSP70 family genes in NIH3T3 cells. LL-Z1640-2 seems to be useful to analyze functions of toxic metal-induced JNK/p38 activation

Oxide Synthase Expression by p38 MAP Kinase

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Tuija Turpeinen

2011-01-01

Full Text Available The role of dual specificity phosphatase 1 (DUSP1 in inducible nitric oxide synthase (iNOS expression in A549 human pulmonary epithelial cells, J774 mouse macrophages and primary mouse bone marrow-derived macrophages (BMMs was investigated. iNOS expression was induced by a cytokine mixture (TNF, IFNγ and IL-1β in A549 cells and by LPS in J774 cells, and it was inhibited by p38 MAPK inhibitors SB202190 and BIRB 796. Stimulation with cytokine mixture or LPS enhanced also DUSP1 expression. Down-regulation of DUSP1 by siRNA increased p38 MAPK phosphorylation and iNOS expression in A549 and J774 cells. In addition, LPS-induced iNOS expression was enhanced in BMMs from DUSP1(−/− mice as compared to that in BMMs from wild-type mice. The results indicate that DUSP1 suppresses iNOS expression by limiting p38 MAPK activity in human and mouse cells. Compounds that enhance DUSP1 expression or modulate its function may be beneficial in diseases complicated with increased iNOS-mediated NO production.

Crucial roles of the protein kinases MK2 and MK3 in a mouse model of glomerulonephritis.

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Adam J Guess

Full Text Available Elevated mitogen-activated protein kinase p38 (p38 MAPK signaling has been implicated in various experimental and human glomerulopathies, and its inhibition has proven beneficial in animal models of these diseases. p38 MAPK signaling is partially mediated through MK2 and MK3, two phylogenetically related protein kinases that are its direct substrates. The current study was designed to determine the specific roles of MK2 and MK3 in a mouse model of acute proliferative glomerulonephritis, using mice with disrupted MK2 and/or MK3 genes. We found that the absence of MK3 alone worsened the disease course and increased mortality slightly compared to wild-type mice, whereas the absence of MK2 alone exhibited no significant effect. However, in an MK3-free background, the disease course depended on the presence of MK2 in a gene dosage-dependent manner, with double knock-out mice being most susceptible to disease induction. Histological and renal functional analyses confirmed kidney damage following disease induction. Because the renal stress response plays a crucial role in kidney physiology and disease, we analyzed the stress response pattern in this disease model. We found that renal cortices of diseased mice exhibited a pronounced and specific pattern of expression and/or phosphorylation of stress proteins and other indicators of the stress response (HSPB1, HSPB6, HSPB8, CHOP, eIF2α, partially in a MK2/MK3 genotype-specific manner, and without induction of a general stress response. Similarly, the expression and activation patterns of other protein kinases downstream of p38 MAPK (MNK1, MSK1 depended partially on the MK2/MK3 genotype in this disease model. In conclusion, MK2 and MK3 together play crucial roles in the regulation of the renal stress response and in the development of glomerulonephritis, which can potentially be exploited to develop novel therapeutic approaches to treat glomerular disease.

PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

Energy Technology Data Exchange (ETDEWEB)

Park, Jeong Su [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kang, Dong Hoon [Department of Life Science and Ewha Research Center for Systems Biology (Korea, Republic of); The Research Center for Cell Homeostasis, Ewha Womans University, Seoul 127-750 (Korea, Republic of); Lee, Da Hyun [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Bae, Soo Han, E-mail: soohanbae@yuhs.ac [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

2015-10-23

p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.

The Histone Deacetylase Inhibitors MS-275 and SAHA Suppress the p38 Mitogen-Activated Protein Kinase Signaling Pathway and Chemotaxis in Rheumatoid Arthritic Synovial Fibroblastic E11 Cells

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Hai-Shu Lin

2013-11-01

Full Text Available MS-275 (entinostat and SAHA (vorinostat, two histone deacetylase (HDAC inhibitors currently in oncological trials, have displayed potent anti-rheumatic activities in rodent models of rheumatoid arthritis (RA. To further elucidate their anti-inflammatory mechanisms, the impact of MS-275 and SAHA on the p38 mitogen-activated protein kinase (MAPK signaling pathway and chemotaxis was assessed in human rheumatoid arthritic synovial fibroblastic E11 cells. MS-275 and SAHA significantly suppressed the expression of p38α  MAPK, but induced the expression of MAPK phosphatase-1 (MKP-1, an endogenous suppressor of p38α  in E11 cells. At the same time, the association between p38α and MKP-1 was up-regulated and consequently, the activation (phosphorylation of p38α  was inhibited. Moreover, MS-275 and SAHA suppressed granulocyte chemotactic protein-2 (GCP-2, monocyte chemotactic protein-2 (MCP-2 and macrophage migration inhibitory factor (MIF in E11 cells in a concentration-dependent manner. Subsequently, E11-driven migration of THP-1 and U937 monocytes was inhibited. In summary, suppression of the p38 MAPK signaling pathway and chemotaxis appear to be important anti-rheumatic mechanisms of action of these HDAC inhibitors.

Amide-based inhibitors of p38alpha MAP kinase. Part 2: design, synthesis and SAR of potent N-pyrimidyl amides.

Science.gov (United States)

Tester, Richland; Tan, Xuefei; Luedtke, Gregory R; Nashashibi, Imad; Schinzel, Kurt; Liang, Weiling; Jung, Joon; Dugar, Sundeep; Liclican, Albert; Tabora, Jocelyn; Levy, Daniel E; Do, Steven

2010-04-15

Optimization of a tri-substituted N-pyridyl amide led to the discovery of a new class of potent N-pyrimidyl amide based p38alpha MAP kinase inhibitors. Initial SAR studies led to the identification of 5-dihydrofuran as an optimal hydrophobic group. Additional side chain modifications resulted in the introduction of hydrogen bond interactions. Through extensive SAR studies, analogs bearing free amino groups and alternatives to the parent (S)-alpha-methyl benzyl moiety were identified. These compounds exhibited improved cellular activities and maintained balance between p38alpha and CYP3A4 inhibition. Copyright 2010 Elsevier Ltd. All rights reserved.

Opposite effects of Ha-Ras and Ki-Ras on radiation-induced apoptosis via differential activation of PI3K/Akt and Rac/p38 mitogen-activated protein kinase signaling

International Nuclear Information System (INIS)

Choi, J.-A.; Kang, C.-M.; Lee, Y.-S.; Lee, S.-J.; Bae, S.-W.; Cho, C.-K.

2003-01-01

It has been well known that Ras signaling is involved in various cellular processes, including proliferation, differentiation, and apoptosis. However, distinct cellular functions of Ras isozymes are not fully understood. Here we show the opposing roles of Ha-Ras and Ki-Ras genes in the modulation of cell sensitivity to ionizing radiation. Overexpression of active isoform of Ha-Ras (12V-Ha- Ras) in Rat2 cells increases resistance to the ionizing radiation. Constitutive activation of phosphoinositide-3-kinase (PI3K) and Akt is detected specifically in 12V-Ha-Ras-overexpressing cells. The specific PI3K inhibitor LY294002 inhibits PI3K/Akt signaling and potentiates the radiation-induced apoptosis, suggesting that activation of PI3K/Akt signaling pathway is involved in the increased radio-resistance in cells overexpressing 12V-Ha-Ras. Overexpression of activated Ki-Ras (12V-Ki-Ras), on the other hand, markedly increases radiation sensitivity. The p38 mitogen-activated protein (MAP) kinase activity is selectively enhanced by ionizing radiation in cells overexpressing 12V-Ki-Ras. The specific p38 MAP kinase inhibitor, PD169316, or dominant-negative p38 MAP kinase decreases radiation-induced cell death. We further show that the mechanism that underlies potentiation of cell death in cells overexpressing 12V-Ki-Ras involves Bax translocation to the mitochondrial membrane. Elevated Bax translocation following ionizing irradiation in 12V-Ki-Ras-overexpressing cells is completely inhibited by PD169316 or dominant-negative p38 MAP kinase. In addition, introduction of cells with RacN17, a dominant negative mutant of Rac, resulted in a marked inhibition of radiation-induced Bax translocation and apoptotic cell death as well as p38 MAP kinase activation. Taken together, these findings explain the opposite effects of Ha-Ras and Ki-Ras on modulation of radio-sensitivity, and suggest that differential activation of PI3K/Akt and Rac/p38 MAP kinase signaling by Ha-Ras and Ki-Ras may

Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

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Defeng Wu

2013-01-01

Full Text Available Autophagy has been shown to be protective against drug and alcohol-induced liver injury. CYP2E1 plays a role in the toxicity of ethanol, carcinogens and certain drugs. Inhibition of autophagy increased ethanol-toxicity and accumulation of fat in wild type and CYP2E1 knockin mice but not in CYP2E1 knockout mice as well as in HepG2 cells expressing CYP2E1 (E47 cells but not HepG2 cells lacking CYP2E1 (C34 cells. The goal of the current study was to evaluate whether modulation of autophagy can affect CYP2E1-dependent cytotoxicity in the E47 cells. The agents used to promote CYP2E1 –dependent toxicity were a polyunsaturated fatty acid, arachidonic acid (AA, buthionine sulfoximine (BSO, which depletes GSH, and CCl4, which is metabolized to the CCl3 radical. These three agents produced a decrease in E47 cell viability which was enhanced upon inhibition of autophagy by 3-methyladenine (3-MA or Atg 7 siRNA. Toxicity was lowered by rapamycin which increased autophagy and was much lower to the C34 cells which do not express CYP2E1. Toxicity was mainly necrotic and was associated with an increase in reactive oxygen production and oxidative stress; 3-MA increased while rapamycin blunted the oxidative stress. The enhanced toxicity and ROS formation produced when autophagy was inhibited was prevented by the antioxidant N-Acetyl cysteine. AA, BSO and CCl4 produced mitochondrial dysfunction, lowered cellular ATP levels and elevated mitochondrial production of ROS. This mitochondrial dysfunction was enhanced by inhibition of autophagy with 3-MA but decreased when autophagy was increased by rapamycin. The mitogen activated protein kinases p38 MAPK and JNK were activated by AA especially when autophagy was inhibited and chemical inhibitors of p38 MAPK and JNK lowered the elevated toxicity of AA produced by 3-MA. These results show that autophagy was protective against the toxicity produced by several agents known to be activated by CYP2E1. Since CYP2E1 plays an

Unbinding Kinetics of a p38 MAP Kinase Type II Inhibitor from Metadynamics Simulations.

Science.gov (United States)

Casasnovas, Rodrigo; Limongelli, Vittorio; Tiwary, Pratyush; Carloni, Paolo; Parrinello, Michele

2017-04-05

Understanding the structural and energetic requisites of ligand binding toward its molecular target is of paramount relevance in drug design. In recent years, atomistic free energy calculations have proven to be a valid tool to complement experiments in characterizing the thermodynamic and kinetic properties of protein/ligand interaction. Here, we investigate, through a recently developed metadynamics-based protocol, the unbinding mechanism of an inhibitor of the pharmacologically relevant target p38 MAP kinase. We provide a thorough description of the ligand unbinding pathway identifying the most stable binding mode and other thermodynamically relevant poses. From our simulations, we estimated the unbinding rate as k off = 0.020 ± 0.011 s -1 . This is in good agreement with the experimental value (k off = 0.14 s -1 ). Next, we developed a Markov state model that allowed identifying the rate-limiting step of the ligand unbinding process. Our calculations further show that the solvation of the ligand and that of the active site play crucial roles in the unbinding process. This study paves the way to investigations on the unbinding dynamics of more complex p38 inhibitors and other pharmacologically relevant inhibitors in general, demonstrating that metadynamics can be a powerful tool in designing new drugs with engineered binding/unbinding kinetics.

Effect of lipopolysaccharide and chlorpromazine on glucocorticoid receptor-mediated gene transcription and immunoreactivity: a possible involvement of p38-MAP kinase

Czech Academy of Sciences Publication Activity Database

Basta-Kaim, A.; Budziszewska, B.; Jaworska-Feil, L.; Tetich, M.; Kubera, M.; Zajícová, Alena; Holáň, Vladimír; Lasoń, W.

2004-01-01

Roč. 14, č. 6 (2004), s. 521-528 ISSN 0924-977X R&D Projects: GA MŠk LN00A026 Institutional research plan: CEZ:AV0Z5052915 Keywords : p38-MAP kinase, cytokines, gene transcription Subject RIV: EC - Immunology Impact factor: 3.545, year: 2004

Uric acid stimulates proliferative pathways in vascular smooth muscle cells through the activation of p38 MAPK, p44/42 MAPK and PDGFRβ.

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Kırça, M; Oğuz, N; Çetin, A; Uzuner, F; Yeşilkaya, A

2017-04-01

Hyperuricemia and angiotensin II (Ang II) may have a pathogenetic role in the development of hypertension and atherosclerosis as well as cardiovascular disease (CVD) and its prognosis. The purpose of this study was to investigate whether uric acid can induce proliferative pathways of vascular smooth muscle cell (VSMC) that are thought to be responsible for the development of CVD. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and platelet-derived growth factor receptor β (PDGFRβ) was measured by Elisa and Western blot techniques to determine the activation of proliferative pathways in primary cultured VSMCs from rat aorta. Results demonstrated that uric acid can stimulate p38 MAPK, p44/42 MAPK and PDGFRβ phosphorylation in a time- and concentration-dependent manner. Furthermore, treatment of VSMCs with the angiotensin II type I receptor (AT1R) inhibitor losartan suppressed p38 MAPK and p44/42 MAPK induction by uric acid. The stimulatory effect of uric acid on p38 MAPK was higher compared to that of Ang II. The results of this study show for the first time that uric acid-induced PDGFRβ phosphorylation plays a crucial role in the development of CVDs and that elevated uric acid levels could be a potential therapeutical target in CVD patients.

In Silico Screening and In Vitro Activity Measurement of Javamide Analogues as Potential p38 MAPK Inhibitors

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Jae B. Park

2017-12-01

Full Text Available p38 Mitogen-activated protein kinase (p38 MAPK is a protein kinase critically involved in the progress of inflammation/stress-associated diseases. Our data suggested that javamide analogues may contain strong anti-inflammation activities, but there is little information about their effects on p38 MAPK. Therefore, in this paper, the effects of thirty javamide analogues on p38 MAPK were investigated using in silico screening and in vitro p38 MAPK assay methods. The javamide analogues were synthesized and their chemical structures were confirmed using nuclear magnetic resonance (NMR spectroscopic methods. Then, the javamide analogues were screened using an in silico modeling program. The screened analogues demonstrated a wide range of binding energy (ΔE; −20 to −39 and several analogues with ΔE; −34 to −39 showed strong binding affinity to p38 MAPK. In vitro p38 MAPK assay, the kinase was significantly inhibited by the analogues with great binding energy (ΔE; −34 to −39 and in silico scores (Avg. score; −27.5 to −29.3. Furthermore, the comparative analysis of both assays showed a positive correlation between the in silico scores and p38 MAPK inhibition. In fact, the javamide analogues with top five in silico scores (Avg. score; −27.5 to −29.3 were found to inhibit p38 MAPK by 27–31% (p < 0.05 better than those with less scores (ΔE < −27.0. Especially, javamide-II-O-ethyl ester with relatively high in silico score (Avg. score; −29.2 inhibited p38 MAPK (IC50 = 9.9 μM a little better than its methyl ester with best in silico score (Avg. score; −29.3. To support the ability to inhibit p38 MAPK, the treatment of javamide-II-ethyl and -methyl esters could suppress the production of IL-8 and MCP-1 protein significantly by 22–73% (p < 0.05 in the differentiated THP-1 cells, and the inhibition was slightly stronger by the ethyl ester than the methyl ester. Altogether, this study suggests that javamide-II-O-ethyl ester may

Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption

DEFF Research Database (Denmark)

Beck, Halfdan; Nähse-Kumpf, Viola; Larsen, Marie Sofie Yoo

2012-01-01

Activation of oncogenes or inhibition of WEE1 kinase deregulates Cyclin-dependent kinase (CDK) activity and leads to replication stress, however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibiting WEE1 kinase rapidly increases initiation of replic......Activation of oncogenes or inhibition of WEE1 kinase deregulates Cyclin-dependent kinase (CDK) activity and leads to replication stress, however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibiting WEE1 kinase rapidly increases initiation...... of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted...

Chronic tooth pulp inflammation induces persistent expression of phosphorylated ERK (pERK) and phosphorylated p38 (pp38) in trigeminal subnucleus caudalis

Science.gov (United States)

Worsley, M.A.; Allen, C.E.; Billinton, A.; King, A.E.; Boissonade, F.M.

2014-01-01

Background Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase are transiently phosphorylated (activated) in the spinal cord and trigeminal nucleus by acute noxious stimuli. Acute stimulation of dental pulp induces short-lived ERK activation in trigeminal subnucleus caudalis (Vc), and p38 inhibition attenuates short-term sensitization in Vc induced by acute pulpal stimulation. We have developed a model to study central changes following chronic inflammation of dental pulp that induces long-term sensitization. Here, we examine the effects of chronic inflammation and acute stimulation on the expression of phosphorylated ERK (pERK), phosphorylated p38 (pp38) and Fos in Vc. Results Chronic inflammation alone induced bilateral expression of pERK and pp38 in Vc, but did not induce Fos expression. Stimulation of both non-inflamed and inflamed pulps significantly increased pERK and pp38 bilaterally; expression was greatest in inflamed, stimulated animals, and was similar following 10-min and 60-min stimulation. Stimulation for 60 min, but not 10 min, induced Fos in ipsilateral Vc; Fos expression was significantly greater in inflamed, stimulated animals. pERK was present in both neurons and astrocytes; pp38 was present in neurons and other non-neuronal, non-astrocytic cell types. Conclusions This study provides the first demonstration that chronic inflammation of tooth pulp induces persistent bilateral activation of ERK and p38 within Vc, and that this activation is further increased by acute stimulation. This altered activity in intracellular signaling is likely to be linked to the sensitization that is seen in our animal model and in patients with pulpitis. Our data indicate that pERK and pp38 are more accurate markers of central change than Fos expression. In our model, localization of pERK and pp38 within specific cell types differs from that seen following acute stimulation. This may indicate specific roles for different cell types in

Hepatitis B Virus Middle Protein Enhances IL-6 Production via p38 MAPK/NF-κB Pathways in an ER Stress-Dependent Manner.

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Yang-Xia Li

Full Text Available During hepatitis B virus (HBV infection, three viral envelope proteins of HBV are overexpressed in the endoplasmic reticulum (ER. The large S protein (LHBs and truncated middle S protein (MHBst have been documented to play roles in regulating host gene expression and contribute to hepatic disease development. As a predominant protein at the ultrastructural level in biopsy samples taken from viremic patients, the role of the middle S protein (MHBs remains to be understood despite its high immunogenicity. When we transfected hepatocytes with an enhanced green fluorescent protein (EGFP-tagged MHBs expressing plasmid, the results showed that expression of MHBs cause an upregulation of IL-6 at the message RNA and protein levels through activating the p38 mitogen-activated protein kinase (p38 MAPK and nuclear factor-kappa B (NF-κB pathways. The use of specific inhibitors of the signaling pathways can diminish this upregulation. The use of BAPTA-AM attenuated the stimulation caused by MHBs. We further found that MHBs accumulated in the endoplasmic reticulum and increased the amount of glucose regulated protein 78 (GRP78/BiP. Our results provide a possibility that MHBs could be involved in liver disease progression.

Neuroprotective effects of ebselen in traumatic brain injury model: involvement of nitric oxide and p38 mitogen-activated protein kinase signalling pathway.

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Wei, Liang; Zhang, Yanfei; Yang, Cheng; Wang, Qi; Zhuang, Zhongwei; Sun, Zhiyang

2014-02-01

Previous investigations have found that ebselen is able to treat neurodegenerative diseases caused by radical and acute total cerebral ischaemia. The aim of the present study was to investigate the neuroprotective effects of ebselen in a traumatic brain injury (TBI) model. Ninety Sprague-Dawley rats were randomly divided into five groups (n = 18 in each): (i) sham operation; (ii) an injury model group; (iii) low-dose (3 mg/kg) ebselen-treated group; (iv) a moderate-dose (10 mg/kg) ebselen-treated group; and (v) a high-dose (30 mg/kg) ebselen-treated group. The TBI model was created according using a modified weight-drop model. Neurological severity score (NSS), brain water content and histopathological deficits were assessed as parameters of injury severity. Expression of nitric oxide (NO), inducible NO synthase (iNOS) mRNA, Toll-like receptor (TLR) and phosphorylated (p-) p38 mitogen-activated protein kinase (MAPK) were examined by chemical colorimetry, quantitative polymerase chain reaction and western blotting 24 h after intragastric ebselen administration. Rats in the TBI model group exhibited markedly more severe neurological injury (higher NSS, more brain water content and more histopathological deficits) than those in the sham-operated group. Ebselen treatment significantly ameliorated the neurological injury of TBI rats in a dose-dependent manner. Moreover, ebselen significantly reduced the NO and iNOS mRNA levels and inhibited TLR4 and p-p38 MAPK expression, indicating the involvement of NO and p38 MAPK signalling pathways in the neuroprotection afforded by ebselen. In conclusion, ebselen ameliorated neurological injury, possibly by reducing NO levels and modulating the TLR4-mediated p38 MAPK signalling pathway. Therefore, ebselen may have potential to treat secondary injuries of TBI. © 2013 Wiley Publishing Asia Pty Ltd.

p38 mitogen-activated protein kinase up-regulates LPS-induced NF-κB activation in the development of lung injury and RAW 264.7 macrophages

International Nuclear Information System (INIS)

Kim, Hee J.; Lee, Hui S.; Chong, Young H.; Kang, Jihee Lee

2006-01-01

Clarification of the key regulatory steps that lead to nuclear factor-kappa B (NF-κB) under cellular and pathological conditions is very important. The action of p38 mitogen-activated protein kinase (MAPK) on the upstream of NF-κB activation remains controversial. To examine this issue using an in vivo lung injury model, SB203580, a p38 MAPK inhibitor was given intraorally 1 h prior to lipopolysaccharide (LPS) treatment (intratracheally). The mice were sacrificed 4 h after LPS treatment. SB203580 substantially suppressed LPS-induced rises in p38 MAPK phosphorylation, neutrophil recruitment, total protein content in bronchoalveolar lavage fluid, and apoptosis of bronchoalveolar cells. Furthermore, SB203580 blocked LPS-induced NF-κB activation in lung tissue through down-regulation of serine phosphorylation, degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that, in cultured RAW 264.7 macrophages, SB203580 also blocked LPS-induced NF-κB activation in a dose-dependent manner. SB203580 inhibited LPS-induced serine phosphorylation, degradation of IκB-α, and tyrosine phosphorylation of p65 NF-κB. These data indicate that p38 MAPK acts upstream of LPS-induced NF-κB activation by modulating the phosphorylation of IκB-α and p65 NF-κB during acute lung injury. Because LPS-stimulated macrophages may contribute to inflammatory lung injury, the inhibition of the p38 MAPK-mediated intracellular signaling pathway leading to NF-κB activation represents a target for the attenuation of lung inflammation and parenchymal damage

Mitigation of direct neurotoxic effects of lidocaine and amitriptyline by inhibition of p38 mitogen-activated protein kinase in vitro and in vivo

NARCIS (Netherlands)

Lirk, Philipp; Haller, Ingrid; Myers, Robert R.; Klimaschewski, Lars; Kau, Yi-Chuan; Hung, Yu-Chun; Gerner, Peter

2006-01-01

Local anesthetic-induced direct neurotoxicity (paresthesia, failure to regain normal sensory and motor function) is a potentially devastating complication of regional anesthesia. Local anesthetics activate the p38 mitogen-activated protein kinase (MAPK) system, which is involved in apoptotic cell

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

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Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

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Laura Graf

2013-12-01

Full Text Available The human cytomegalovirus (HCMV-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.

Cadmium induces apoptosis in pancreatic β-cells through a mitochondria-dependent pathway: the role of oxidative stress-mediated c-Jun N-terminal kinase activation.

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Kai-Chih Chang

Full Text Available Cadmium (Cd, one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM. However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic β-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic β-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS generation and malondialdehyde (MDA production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP and the increase of cytosolic cytochrome c release, the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose polymerase (PARP cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK, extracellular signal-regulated kinases (ERK1/2, and p38-mitogen-activated protein kinase (MAPK, which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA transfection suppressed Cd-induced β-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580 did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic β-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway.

Expression and proliferation profiles of PKC, JNK and p38MAPK in physiologically stretched human bladder smooth muscle cells

International Nuclear Information System (INIS)

Wazir, Romel; Luo, De-Yi; Dai, Yi; Yue, Xuan; Tian, Ye; Wang, Kun-Jie

2013-01-01

Highlights: •Stretch induces proliferation in human bladder smooth muscle cells (HBSMC). •5% Equibiaxial elongation produces maximum proliferation. •Physiologic stretch decreases apoptotic cell death. •PKC is involved in functional modulation of bladder. •JNK and p38 are not involved in proliferating HBSMC. -- Abstract: Objective: To determine protein kinase C (PKC), c-Jun NH2-Terminal Kinase (JNK) and P38 mitogen-activated protein kinases (p38MAPK) expression levels and effects of their respective inhibitors on proliferation of human bladder smooth muscle cells (HBSMCs) when physiologically stretched in vitro. Materials and methods: HBSMCs were grown on silicone membrane and stretch was applied under varying conditions; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (frequency: 0.05, 0.1, 0.2, 0.5, 1 Hz). Optimal physiological stretch was established by assessing proliferation with 5-Bromo-2-deoxyuridine (BrdU) assay and flow cytometry. PKC, JNK and p38 expression levels were analyzed by Western blot. Specificity was maintained by employing specific inhibitors; (GF109203X for PKC, SP600125 for JNK and SB203580 for p38MAPK), in some experiments. Results: Optimum proliferation was observed at 5% equibiaxial stretch (BrdU: 0.837 ± 0.026 (control) to 1.462 ± 0.023)%, (P 0.05 SP600125) and (1.461 ± 0.01, P > 0.05 SB203580). These findings show that mechanical stretch can promote magnitude-dependent proliferative modulation through PKC and possibly JNK but not via p38MAPK in hBSMCs

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

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Serena A D'Souza

2016-04-01

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

Induction of apoptosis in renal cell carcinoma by reactive oxygen species: involvement of extracellular signal-regulated kinase 1/2, p38delta/gamma, cyclooxygenase-2 down-regulation, and translocation of apoptosis-inducing factor.

LENUS (Irish Health Repository)

Ambrose, Monica

2012-02-03

Renal cell carcinoma (RCC) is the most common malignancy of the kidney. Unfortunately, RCCs are highly refractory to conventional chemotherapy, radiation therapy, and even immunotherapy. Thus, novel therapeutic targets need to be sought for the successful treatment of RCCs. We now report that 6-anilino-5,8-quinolinequinone (LY83583), an inhibitor of cyclic GMP production, induced growth arrest and apoptosis of the RCC cell line 786-0. It did not prove deleterious to normal renal epithelial cells, an important aspect of chemotherapy. To address the cellular mechanism(s), we used both genetic and pharmacological approaches. LY83583 induced a time- and dose-dependent increase in RCC apoptosis through dephosphorylation of mitogen-activated protein kinase kinase 1\\/2 and its downstream extracellular signal-regulated kinases (ERK) 1 and -2. In addition, we observed a decrease in Elk-1 phosphorylation and cyclooxygenase-2 (COX-2) down-regulation. We were surprised that we failed to observe an increase in either c-Jun NH(2)-terminal kinase or p38alpha and -beta mitogen-activated protein kinase activation. In contradiction, reintroduction of p38delta by stable transfection or overexpression of p38gamma dominant negative abrogated the apoptotic effect. Cell death was associated with a decrease and increase in Bcl-x(L) and Bax expression, respectively, as well as release of cytochrome c and translocation of apoptosis-inducing factor. These events were associated with an increase in reactive oxygen species formation. The antioxidant N-acetyl l-cysteine, however, opposed LY83583-mediated mitochondrial dysfunction, ERK1\\/2 inactivation, COX-2 down-regulation, and apoptosis. In conclusion, our results suggest that LY83583 may represent a novel therapeutic agent for the treatment of RCC, which remains highly refractory to antineoplastic agents. Our data provide a molecular basis for the anticancer activity of LY83583.

Poxviral protein A52 stimulates p38 mitogen-activated protein kinase (MAPK) activation by causing tumor necrosis factor receptor-associated factor 6 (TRAF6) self-association leading to transforming growth factor β-activated kinase 1 (TAK1) recruitment.

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Stack, Julianne; Hurst, Tara P; Flannery, Sinead M; Brennan, Kiva; Rupp, Sebastian; Oda, Shun-ichiro; Khan, Amir R; Bowie, Andrew G

2013-11-22

Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation.

Mycobacterium tuberculosis-infected human monocytes down-regulate microglial MMP-2 secretion in CNS tuberculosis via TNFα, NFκB, p38 and caspase 8 dependent pathways

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Elkington Paul T

2011-05-01

Full Text Available Abstract Tuberculosis (TB of the central nervous system (CNS is a deadly disease characterized by extensive tissue destruction, driven by molecules such as Matrix Metalloproteinase-2 (MMP-2 which targets CNS-specific substrates. In a simplified cellular model of CNS TB, we demonstrated that conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb, but not direct infection, unexpectedly down-regulates constitutive microglial MMP-2 gene expression and secretion by 72.8% at 24 hours, sustained up to 96 hours (P M.tb-infected monocyte-dependent networks paradoxically involves the pro-inflammatory mediators TNF-α, p38 MAP kinase and NFκB in addition to a novel caspase 8-dependent pathway.

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

International Nuclear Information System (INIS)

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

2008-01-01

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

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

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Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

CIKS, a connection to IκB kinase and stress-activated protein kinase

Science.gov (United States)

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

EGFR-dependent signalling reduced and p38 dependent apoptosis required by Gallic acid in Malignant Mesothelioma cells.

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Demiroglu-Zergeroglu, Asuman; Candemir, Gulsife; Turhanlar, Ebru; Sagir, Fatma; Ayvali, Nurettin

2016-12-01

The unrestrained EGFR signalling contributes to malignant phenotype in a number of cancers including Malignant Mesotheliomas. Present study was designed to evaluate EGFR-dependent anti-proliferative and apoptotic effects of Gallic acid in transformed Mesothelial (MeT-5A) and Malignant Mesothelioma (SPC212) cells. Gallic acid reduced the viability of Malignant Mesothelioma cells in a concentration and time-dependent manner. However, viability of mesothelial cells reduced only at high concentration and longer time periods. Gallic acid restrained the activation of EGFR, ERK1/2 and AKT proteins and down regulated expression of Cyclin D and Bcl-2 genes, but upregulated the expression of p21 gene in EGF-induced SPC212 cells. GA-induced transitory G1 arrest and triggered mitochondrial and death receptor mediated apoptosis, which requires p38MAPK activation. The data provided here indicate that GA is able to inhibit EGFR dependent proliferation and survival signals and induces p38 pathway dependent apoptosis in Malignant Mesothelioma cells. On the basis of these experimental findings it is worthwhile to investigate further the biological activity of Gallic acid on other Mesothelioma cell lines harbouring aberrant EGFR signals. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Evaluating the Role of p38 MAPK in the Accelerated Cell Senescence of Werner Syndrome Fibroblasts

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Terence Davis

2016-04-01

Full Text Available Progeroid syndromes show features of accelerated ageing and are used as models for human ageing, of which Werner syndrome (WS is one of the most widely studied. WS fibroblasts show accelerated senescence that may result from p38 MAP kinase activation since it is prevented by the p38 inhibitor SB203580. Thus, small molecule inhibition of p38-signalling may be a therapeutic strategy for WS. To develop this approach issues such as the in vivo toxicity and kinase selectivity of existing p38 inhibitors need to be addressed, so as to strengthen the evidence that p38 itself plays a critical role in mediating the effect of SB203580, and to find an inhibitor suitable for in vivo use. In this work we used a panel of different p38 inhibitors selected for: (1 having been used successfully in vivo in either animal models or human clinical trials; (2 different modes of binding to p38; and (3 different off-target kinase specificity profiles, in order to critically address the role of p38 in the premature senescence seen in WS cells. Our findings confirmed the involvement of p38 in accelerated cell senescence and identified p38 inhibitors suitable for in vivo use in WS, with BIRB 796 the most effective.

c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

International Nuclear Information System (INIS)

Sprowles, Amy; Robinson, Dan; Wu Yimi; Kung, H.-J.; Wisdom, Ron

2005-01-01

The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis to define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli

Protective effect of tropisetron on rodent hepatic injury after trauma-hemorrhagic shock through P38 MAPK-dependent hemeoxygenase-1 expression.

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Fu-Chao Liu

Full Text Available Tropisetron can decrease inflammatory cell responses and alleviate organ damage caused by trauma-hemorrhage, but the mechanism of these effects remains unknown. The p38 mitogen-activated protein kinase/hemeoxygenase-1 (p38 MAPK/HO-1 pathway exerts anti-inflammatory effects on different tissues. The aim of this study was to investigate whether p38 MAPK/HO-1 plays any role in the tropisetron-mediated attenuation of hepatic injury after trauma-hemorrhage. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure maintained at approximately 35-40 mmHg for 90 min, followed by fluid resuscitation. During resuscitation, several treatment regimens were administered: four doses of tropisetron alone (0.1, 0.3, 1, 3 mg/kg body weight, or a single dose of tropisetron (1 mg/kg body weight with and without a p38 MAPK inhibitor (SB-203580, 2 mg/kg body weight or HO antagonist (chromium-mesoporphyrin, 2.5 mg/kg body weight. Various parameters were measured, and the animals were sacrificed at 24 h post-resuscitation. The results showed that trauma-hemorrhage increased the following parameters: plasma concentrations of aspartate (AST and alanine aminotransferases (ALT, hepatic myeloperoxidase (MPO activity, and levels of cytokine-induced neutrophil chemoattractant-1 and -3 (CINC-1 and CINC-3, intercellular adhesion molecule-1 (ICAM-1, interleukin-6 (IL-6, tumor necrosis factor-α (TNF-α, and macrophage inflammatory protein-1α (MIP-1α. These parameters were significantly improved in the tropisetron-treated rats subjected to trauma-hemorrhage. Tropisetron treatment also increased hepatic p38 MAPK and HO-1 expression compared with vehicle-treated trauma-hemorrhaged rats. Co-administration of SB-203580 or chromium-mesoporphyrin with tropisetron abolished the tropisetron-induced beneficial effects on the above parameters and hepatic injury. These results suggest that the protective effect of tropisetron administration on alleviation of hepatic

Protective effect of tropisetron on rodent hepatic injury after trauma-hemorrhagic shock through P38 MAPK-dependent hemeoxygenase-1 expression.

Science.gov (United States)

Liu, Fu-Chao; Yu, Huang-Ping; Hwang, Tsong-Long; Tsai, Yung-Fong

2012-01-01

Tropisetron can decrease inflammatory cell responses and alleviate organ damage caused by trauma-hemorrhage, but the mechanism of these effects remains unknown. The p38 mitogen-activated protein kinase/hemeoxygenase-1 (p38 MAPK/HO-1) pathway exerts anti-inflammatory effects on different tissues. The aim of this study was to investigate whether p38 MAPK/HO-1 plays any role in the tropisetron-mediated attenuation of hepatic injury after trauma-hemorrhage. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure maintained at approximately 35-40 mmHg for 90 min), followed by fluid resuscitation. During resuscitation, several treatment regimens were administered: four doses of tropisetron alone (0.1, 0.3, 1, 3 mg/kg body weight), or a single dose of tropisetron (1 mg/kg body weight) with and without a p38 MAPK inhibitor (SB-203580, 2 mg/kg body weight) or HO antagonist (chromium-mesoporphyrin, 2.5 mg/kg body weight). Various parameters were measured, and the animals were sacrificed at 24 h post-resuscitation. The results showed that trauma-hemorrhage increased the following parameters: plasma concentrations of aspartate (AST) and alanine aminotransferases (ALT), hepatic myeloperoxidase (MPO) activity, and levels of cytokine-induced neutrophil chemoattractant-1 and -3 (CINC-1 and CINC-3), intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein-1α (MIP-1α). These parameters were significantly improved in the tropisetron-treated rats subjected to trauma-hemorrhage. Tropisetron treatment also increased hepatic p38 MAPK and HO-1 expression compared with vehicle-treated trauma-hemorrhaged rats. Co-administration of SB-203580 or chromium-mesoporphyrin with tropisetron abolished the tropisetron-induced beneficial effects on the above parameters and hepatic injury. These results suggest that the protective effect of tropisetron administration on alleviation of hepatic injury

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

DEFF Research Database (Denmark)

Borisova, Marina E; Voigt, Andrea; Tollenaere, Maxim A X

2018-01-01

quantitative phosphoproteomics and protein kinase inhibition to provide a systems view on protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on the canonical DNA damage signaling by ATM/ATR and the p38 MAP kinase pathway. We identify RNA-binding proteins......Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in the DNA that globally affect transcription and splicing. However, the signaling pathways and mechanisms that link UV-light-induced DNA damage to changes in RNA metabolism remain poorly understood. Here we employ...

Suppression of Heregulin-β1/HER2-Modulated Invasive and Aggressive Phenotype of Breast Carcinoma by Pterostilbene via Inhibition of Matrix Metalloproteinase-9, p38 Kinase Cascade and Akt Activation

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Min-Hsiung Pan

2011-01-01

Full Text Available Invasive breast cancer is the major cause of death among females and its incidence is closely linked to HER2 (human epidermal growth factor receptor 2 overexpression. Pterostilbene, a natural analog of resveratrol, exerts its cancer chemopreventive activity similar to resveratrol by inhibiting cancer cell proliferation and inducing apoptosis. However, the anti-invasive effect of pterostilbene on HER2-bearing breast cancer has not been evaluated. Here, we used heregulin-β1 (HRG-β1, a ligand for HER3, to transactivate HER2 signaling. We found that pterostilbene was able to suppress HRG-β1-mediated cell invasion, motility and cell transformation of MCF-7 human breast carcinoma through down-regulation of matrix metalloproteinase-9 (MMP-9 activity and growth inhibition. In parallel, pterostilbene also inhibited protein and mRNA expression of MMP-9 driven by HRG-β1, suggesting that pterostilbene decreased HRG-β1-mediated MMP-9 induction via transcriptional regulation. Examining the signaling pathways responsible for HRG-β1-associated MMP-9 induction and growth inhibition, we observed that pterostilbene, as well as SB203580 (p38 kinase inhibitor, can abolish the phosphorylation of p38 mitogen-activated protein kinase (p38 kinase, a downstream HRG-β1-responsive kinase responsible for MMP-9 induction. In addition, HRG-β1-driven Akt phosphorylation required for cell proliferation was also suppressed by pterostilbene. Taken together, our present results suggest that pterostilbene may serve as a chemopreventive agent to inhibit HRG-β1/HER2-mediated aggressive and invasive phenotype of breast carcinoma through down-regulation of MMP-9, p38 kinase and Akt activation.

Acrolein induces cyclooxygenase-2 and prostaglandin production in human umbilical vein endothelial cells: roles of p38 MAP kinase.

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Park, Yong Seek; Kim, Jayoung; Misonou, Yoshiko; Takamiya, Rina; Takahashi, Motoko; Freeman, Michael R; Taniguchi, Naoyuki

2007-06-01

Acrolein, a known toxin in tobacco smoke, might be involved in atherogenesis. This study examined the effect of acrolein on expression of cyclooxygenase-2 (COX-2) and prostaglandin (PG) production in endothelial cells. Cyclooxygenase (COX)-2 induction by acrolein and signal pathways were measured using Western blots, Northern blots, immunofluorescence, ELISA, gene silencing, and promoter assay. Colocalization of COX2 and acrolein-adduct was determined by immunohistochemistry. Here we report that the levels of COX-2 mRNA and protein are increased in human umbilical vein endothelial cells (HUVECs) after acrolein exposure. COX-2 was found to colocalize with acrolein-lysine adducts in human atherosclerotic lesions. Inhibition of p38 MAPK activity abolished the induction of COX-2 protein and PGE2 accumulation by acrolein, while suppression of extracellular signal-regulated kinase (ERK) and JNK activity had no effect on the induction of COX-2 expression in experiments using inhibitors and siRNA. Furthermore, rottlerin, an inhibitor of protein kinase Cdelta (PKCdelta), abrogated the upregulation of COX-2 at both protein and mRNA levels. These results provide that acrolein may play a role in progression of atherosclerosis and new information on the signaling pathways involved in COX-2 upregulation in response to acrolein and provide evidence that PKCdelta and p38 MAPK are required for transcriptional activation of COX-2.

The expression of cyclin-dependent kinase inhibitors p15, p16, p21, and p27 during ovarian follicle growth initiation in the mouse

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Bayrak Aykut

2003-05-01

Full Text Available Abstract Background Cyclins regulate the cell cycle in association with cyclin dependent kinases (CDKs. CDKs are under inhibitory control of cyclin dependent kinase inhibitors (CDKIs. Method In this study we tested the expression of CDKIs p15, p16, p21 and p27 by immunohistochemistry to determine the role of CDKIs in the initiation of primordial follicle growth. Ovaries were collected from 60-day-old cycling B6D2F1/J mice (n = 16. Results Expression of p15, p16, p21 and p27 did not vary in granulosa and theca cells by the follicle stage. However, p16 staining was stronger (++ in the oocytes of all primordial, and 57.4 ± 3.1% of primary follicles compared to the remaining primary and more advanced follicles (+. Interestingly, primary follicles with weaker (+ oocyte staining for p16 had significantly larger mean follicle diameter compared to the primary and primordial follicles with stronger (++ oocyte staining (55.6 ± 2.1 vs. 32.0 ± 1.0 and 26.5 ± 0.7 μm, respectively, p Conclusions These preliminary findings suggest that the initiation of oocyte growth, which seems to lead follicle growth, is associated with diminished p16 expression in the mouse ovary. Further studies are needed to investigate the factors that regulate the expression of p16 in the oocyte, which might also govern the initiation of primordial follicle growth.

Ebselen suppresses inflammation induced by Helicobacter pylori lipopolysaccharide via the p38 mitogen-activated protein kinase signaling pathway.

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Xu, Ling; Gong, Changguo; Li, Guangming; Wei, Jue; Wang, Ting; Meng, Wenying; Shi, Min; Wang, Yugang

2018-05-01

Ebselen is a seleno-organic compound that has been demonstrated to have antioxidant and anti-inflammatory properties. A previous study determined that ebselen inhibits airway inflammation induced by inhalational lipopolysaccharide (LPS), however, the underlying molecular mechanism remains to be elucidated. The present study investigated the effect of ebselen on the glutathione peroxidase (GPX)‑reactive oxygen species (ROS) pathway and interleukin‑8 (IL‑8) expression induced by Helicobacter pylori LPS in gastric cancer (GC) cells. Cells were treated with 200 ng/ml H. pylori‑LPS in the presence or absence of ebselen for various durations and concentrations (µmol/l). The expression of toll‑like receptor 4 (TLR4), GPX2, GPX4, p38 mitogen‑activated protein kinase (p38 MAPK), phosphorylated‑p38 MAPK, ROS production and IL‑8 expression were detected with western blotting or ELISA. The present study revealed that TLR4 expression was upregulated; however, GPX2 and GPX4 expression was reduced following treatment with H. pylori LPS, which led to increased ROS production, subsequently altering the IL‑8 expression level in GC cells. Additionally, it was determined that ebselen prevented the reduction in GPX2/4 levels induced by H. pylori LPS, however, TLR4 expression was not affected. Ebselen may also block the expression of IL‑8 by inhibiting phosphorylation of p38 MAPK. These data suggest ebselen may inhibit ROS production triggered by H. pylori LPS treatment via GPX2/4 instead of TLR4 signaling and reduce phosphorylation of p38 MAPK, resulting in altered production of IL‑8. Ebselen may, therefore, be a potential therapeutic agent to mediate H. pylori LPS-induced cell damage.

Effects of interleukins 2 and 12 on TBT-induced alterations of MAP kinases p38 and p44/42 in human natural killer cells.

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Aluoch, Aloice O; Whalen, Margaret M

2006-01-01

NK cells are lymphocytes in the non-adaptive immune system that protect the body against intracellular pathogens and eliminate tumor cells. Tributyltin (TBT) is a toxic chemical that has been detected in human foods as well as in human blood. The role of TBT in immunosuppression has been described, including inhibition of the human NK-cell cytotoxic function. Previous studies indicated that exposure of NK cells to TBT for 1 h induced progressive and irreversible inhibition of cytotoxic function. However, it was found that if NK cells were incubated in TBT-free media with either IL-2 or IL-12, loss of cytotoxic function was prevented/reversed within 24 h. Molecular studies established that loss of cytotoxic function is accompanied by alteration of MAP kinases (MAPKs) p38 and p44/42 phosphorylation. This study examined whether interleukin-mediated recovery of cytotoxicity involved reversal of tributyltin-altered p38 and p44/42 phosphorylation. The results indicated that there was no substantial IL-2 prevention/reversal of the TBT-induced alteration of phosphorylation of either p38 or p44/42 after either a 24 or 48 h recovery period. Additionally, IL-12 caused no substantial prevention/reversal of the TBT-induced alteration of phosphorylation of the MAPKs seen after either 24 or 48 h. These data suggest that IL-2 and/or IL-12-mediated recovery of NK cytotoxic function is not a result of prevention/reversal of TBT-induced phosphorylation of p38 and p44/42 MAPKs at the 24 or 48 h time points. Copyright 2005 John Wiley & Sons, Ltd.

Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38MAPK, and JNK Kinase Pathways

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Nicoletta Marchesi

2018-01-01

Full Text Available RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD. HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR’s role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.

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

International Nuclear Information System (INIS)

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

2013-01-01

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

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

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Li, Yanyan; Gao, Chao; Shi, Yanru; Tang, Yuhan; Liu, Liang; Xiong, Ting; Du, Min [Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Ministry of Education Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Xing, Mingyou [Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Liu, Liegang [Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Ministry of Education Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Yao, Ping, E-mail: yaoping@mails.tjmu.edu.cn [Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Ministry of Education Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China)

2013-11-15

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

The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

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Baril, Caroline; Sahmi, Malha; Ashton-Beaucage, Dariel; Stronach, Beth; Therrien, Marc

2009-02-01

The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

Andrographolide induces vascular smooth muscle cell apoptosis through a SHP-1-PP2A-p38MAPK-p53 cascade.

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Chen, Yu-Ying; Hsieh, Cheng-Ying; Jayakumar, Thanasekaran; Lin, Kuan-Hung; Chou, Duen-Suey; Lu, Wan-Jung; Hsu, Ming-Jen; Sheu, Joen-Rong

2014-07-10

The abnormal growth of vascular smooth muscle cells (VSMCs) is considered a critical pathogenic process in inflammatory vascular diseases. We have previously demonstrated that protein phosphatase 2 A (PP2A)-mediated NF-κB dephosphorylation contributes to the anti-inflammatory properties of andrographolide, a novel NF-κB inhibitor. In this study, we investigated whether andrographolide causes apoptosis, and characterized its apoptotic mechanisms in rat VSMCs. Andrographolide activated the p38 mitogen-activated protein kinase (p38MAPK), leading to p53 phosphorylation. Phosphorylated p53 subsequently transactivated the expression of Bax, a pro-apoptotic protein. Transfection with pp2a small interfering RNA (siRNA) suppressed andrographolide-induced p38MAPK activation, p53 phosphorylation, and caspase 3 activation. Andrographolide also activated the Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1), and induced PP2A dephosphorylation, both of which were inhibited by the SHP-1 inhibitor sodium stibogluconate (SSG) or shp-1 siRNA. SSG or shp-1 siRNA prevented andrographolide-induced apoptosis. These results suggest that andrographolide activates the PP2A-p38MAPK-p53-Bax cascade, causing mitochondrial dysfunction and VSMC death through an SHP-1-dependent mechanism.

p38 MAPK mediated in compressive stress-induced chondrogenesis of rat bone marrow MSCs in 3D alginate scaffolds.

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Li, Juan; Zhao, Zhihe; Yang, Jingyuan; Liu, Jun; Wang, Jun; Li, Xiaoyu; Liu, Yurong

2009-12-01

Mesenchymal stem cells (MSCs) are well known to have the capability to form bone and cartilage, and chondrogenesis derived from MSCs is reported to be affected by mechanical stimuli. This research was aimed to study the effects of cyclic compressive stress on the chondrogenic differentiation of rat bone marrow-derived MSCs (BMSCs) which were encapsulated in alginate scaffolds and cultured with or without chondrogenic medium, and to investigate the role of p38 MAPK phospho-relay cascade in this process. The results show that the gene expression of chondrocyte-specific markers of Col2alpha1, aggrecan, Sox9, Runx2, and Ihh was upregulated by dynamic compressive stress introduced at the 8th day of chondrogenic differentiation in vitro. The p38 MAPK was activated by chondrogenic cytokines in a slow and lagged way, but activated by cyclic compressive stimulation in a rapid and transient manner. And inhibition of p38 activity with SB203580 suppressed gene expression of chondrocyte-specific genes stimulated by chondrogenic medium and (or) cyclic compressive stress. These findings suggest that p38 MAPK signal acts as an essential mediator in the mechano-biochemical transduction and subsequent transcriptional regulation in the process of chondrogenesis.

p38 MAPK inhibitors: a patent review (2012 - 2013).

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Bühler, Stefanie; Laufer, Stefan A

2014-05-01

The p38 MAPK is a ubiquitous target in the research-based pharmaceutical industry. It plays a decisive role in the regulation of the production of proinflammatory cytokines. Since novel biological therapies have revolutionized the treatment of chronic inflammatory diseases, an intensive global search is underway for small molecules for the same application. Herein, the patents and the corresponding publications of international companies, which focus on the development and identification of a new generation of small-molecule p38 inhibitors, are summarized. The most promising approach is the development of linear binders, which induce a glycine flip at Gly110 of the kinase hinge region by a carbonyl oxygen atom of the respective ligand. The major focus of the patent works was the application of molecules in new indications. Previous applications were in the treatment of rheumatoid arthritis; currently, there are several new applications, including pulmonary diseases, cancer and Alzheimer's disease. Targeting p38 upstream kinases and downstream effectors has also proved to be a very promising step in the development of more effective inhibitors. A further trend is drug combination, applied to a wide range of indications, such as chronic obstructive pulmonary disease and cancer.

Klotho Protects Dopaminergic Neuron Oxidant-Induced Degeneration by Modulating ASK1 and p38 MAPK Signaling Pathways.

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Reynolds K Brobey

Full Text Available Klotho transgenic mice exhibit resistance to oxidative stress as measured by their urinal levels of 8-hydroxy-2-deoxyguanosine, albeit this anti-oxidant defense mechanism has not been locally investigated in the brain. Here, we tested the hypothesis that the reactive oxygen species (ROS-sensitive apoptosis signal-regulating kinase 1 (ASK1/p38 MAPK pathway regulates stress levels in the brain of these mice and showed that: 1 the ratio of free ASK1 to thioredoxin (Trx-bound ASK1 is relatively lower in the transgenic brain whereas the reverse is true for the Klotho knockout mice; 2 the reduced p38 activation level in the transgene corresponds to higher level of ASK1-bound Trx, while the KO mice showed elevated p38 activation and lower level of-bound Trx; and 3 that 14-3-3ζ is hyper phosphorylated (Ser-58 in the transgene which correlated with increased monomer forms. In addition, we evaluated the in vivo robustness of the protection by challenging the brains of Klotho transgenic mice with a neurotoxin, MPTP and analyzed for residual neuron numbers and integrity in the substantia nigra pars compacta. Our results show that Klotho overexpression significantly protects dopaminergic neurons against oxidative damage, partly by modulating p38 MAPK activation level. Our data highlight the importance of ASK1/p38 MAPK pathway in the brain and identify Klotho as a possible anti-oxidant effector.

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

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Xiaofei Cheng

2016-01-01

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

P38/TRHr-Dependent Regulation of TPO in Thyroid Cells Contributes to the Hypothyroidism of Triclosan-Treated Rats

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Pei Zhang

2018-02-01

Full Text Available Background/Aims: Triclosan, as an antimicrobial agent and a potential endocrine disruptor, has been used extensively in diverse products, resulting in widespread human exposure. In recent years, studies suggest that triclosan could disturb thyroid functions and decline thyroid hormones (THs. Methods: To verify our hypothesis that the MAPK pathway may function significantly in triclosan-induced hypothyroidism, Sprague-Dawley rats were gavaged with triclosan for 31 consecutive days; Nthy-ori 3-1 cells were treated with triclosan in the presence/absence of NAC, inhibitors (SB203580 and SB202474, or TRHr siRNA. Tissues and/or cells were analyzed by several techniques including transmission electron microscopy, confocal laser scanning microscopy, gene silencing, western blot, and real-time PCR. Results: Triclosan led to histopathologic changes in the thyroid and decreases in triiodothyronine (T3 and thyroxine (T4. Triclosan stimulated ROS production and oxidative stress occurrence, thereby activating the p38 pathway in vivo and in vitro. Thyrotropin releasing hormone receptor (TRHr was induced when the p38 pathway was activated, and was suppressed when that pathway was inhibited. Moreover, thyroid peroxidase (TPO was restrained and modulated by the p38/TRHr pathway after triclosan treatment. Furthermore, deiodinase 3 (D3 and hepatic enzymes (Ugt2b1, CYP1a1, CYP1a2, CYP2b1, CYP3a1, and Sult1e1 were also induced by triclosan. Conclusion: Taken together, p38/TRHr-dependent regulation of TPO in thyroid cells contributes to the hypothyroidism of triclosan-treated rats.

Cholesterol Perturbation in Mice Results in p53 Degradation and Axonal Pathology through p38 MAPK and Mdm2 Activation.

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Qingyu Qin

Full Text Available Perturbation of lipid metabolism, especially of cholesterol homeostasis, can be catastrophic to mammalian brain, as it has the highest level of cholesterol in the body. This notion is best illustrated by the severe progressive neurodegeneration in Niemann-Pick Type C (NPC disease, one of the lysosomal storage diseases, caused by mutations in the NPC1 or NPC2 gene. In this study, we found that growth cone collapse induced by genetic or pharmacological disruption of cholesterol egress from late endosomes/lysosomes was directly related to a decrease in axonal and growth cone levels of the phosphorylated form of the tumor suppressor factor p53. Cholesterol perturbation-induced growth cone collapse and decrease in phosphorylated p53 were reduced by inhibition of p38 mitogen-activated protein kinase (MAPK and murine double minute (Mdm2 E3 ligase. Growth cone collapse induced by genetic (npc1-/- or pharmacological modification of cholesterol metabolism was Rho kinase (ROCK-dependent and associated with increased RhoA protein synthesis; both processes were significantly reduced by P38 MAPK or Mdm2 inhibition. Finally, in vivo ROCK inhibition significantly increased phosphorylated p53 levels and neurofilaments in axons, and axonal bundle size in npc1-/- mice. These results indicate that NPC-related and cholesterol perturbation-induced axonal pathology is associated with an abnormal signaling pathway consisting in p38 MAPK activation leading to Mdm2-mediated p53 degradation, followed by ROCK activation. These results also suggest new targets for pharmacological treatment of NPC disease and other diseases associated with disruption of cholesterol metabolism.

Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38α MAP kinase: a combined computational and experimental study.

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Zhu, Shun; Travis, Sue M; Elcock, Adrian H

2013-07-09

A major current challenge for drug design efforts focused on protein kinases is the development of drug resistance caused by spontaneous mutations in the kinase catalytic domain. The ubiquity of this problem means that it would be advantageous to develop fast, effective computational methods that could be used to determine the effects of potential resistance-causing mutations before they arise in a clinical setting. With this long-term goal in mind, we have conducted a combined experimental and computational study of the thermodynamic effects of active-site mutations on a well-characterized and high-affinity interaction between a protein kinase and a small-molecule inhibitor. Specifically, we developed a fluorescence-based assay to measure the binding free energy of the small-molecule inhibitor, SB203580, to the p38α MAP kinase and used it measure the inhibitor's affinity for five different kinase mutants involving two residues (Val38 and Ala51) that contact the inhibitor in the crystal structure of the inhibitor-kinase complex. We then conducted long, explicit-solvent thermodynamic integration (TI) simulations in an attempt to reproduce the experimental relative binding affinities of the inhibitor for the five mutants; in total, a combined simulation time of 18.5 μs was obtained. Two widely used force fields - OPLS-AA/L and Amber ff99SB-ILDN - were tested in the TI simulations. Both force fields produced excellent agreement with experiment for three of the five mutants; simulations performed with the OPLS-AA/L force field, however, produced qualitatively incorrect results for the constructs that contained an A51V mutation. Interestingly, the discrepancies with the OPLS-AA/L force field could be rectified by the imposition of position restraints on the atoms of the protein backbone and the inhibitor without destroying the agreement for other mutations; the ability to reproduce experiment depended, however, upon the strength of the restraints' force constant

A role for barley calcium-dependent protein kinase CPK2a in the response to drought

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Agata Cieśla

2016-10-01

Full Text Available Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L., one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index, an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.

Nephroprotective Effects of N-Acetylcysteine Amide against Contrast-Induced Nephropathy through Upregulating Thioredoxin-1, Inhibiting ASK1/p38MAPK Pathway, and Suppressing Oxidative Stress and Apoptosis in Rats

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Xuezhong Gong

2016-01-01

Full Text Available Contrast-induced nephropathy (CIN is a leading cause of hospital-acquired acute kidney injury (AKI due to apoptosis induced in renal tubular cells. Our previous study demonstrated the novel N-acetylcysteine amide (NACA; the amide form of N-acetyl cysteine (NAC prevented renal tubular cells from contrast-induced apoptosis through inhibiting p38 MAPK pathway in vitro. In the present study, we aimed to compare the efficacies of NACA and NAC in preventing CIN in a well-established rat model and investigate whether thioredoxin-1 (Trx1 and apoptosis signal-regulating kinase 1 (ASK1 act as the potential activator for p38 MAPK. NACA significantly attenuated elevations of serum creatinine, blood urea nitrogen, and biomarkers of AKI. At equimolar concentration, NACA was more effective than NAC in reducing histological changes of renal tubular injuries. NACA attenuated activation of p38 MAPK signal, reduced oxidative stress, and diminished apoptosis. Furthermore, we demonstrated that contrast exposure resulted in Trx1 downregulation and increased ASK1/p38 MAPK phosphorylation, which could be reversed by NACA and NAC. To our knowledge, this is the first report that Trx1 and ASK1 are involved in CIN. Our study highlights a renal protective role of NACA against CIN through modulating Trx1 and ASK1/p38 MAPK pathway to result in the inhibition of apoptosis among renal cells.

Δ8-Tetrahydrocannabinol induces cytotoxicity in macrophage J774-1 cells: Involvement of cannabinoid receptor 2 and p38 MAPK

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Yamaori, Satoshi; Ishii, Hirosuke; Chiba, Kenzo; Yamamoto, Ikuo; Watanabe, Kazuhito

2013-01-01

Tetrahydrocannabinol (THC), a psychoactive component of marijuana, is known to exert cytotoxicity in immune cells. In the present study, we examined the cytotoxicity of Δ 8 -THC in mouse macrophage J774-1 cells and a possible involvement of cannabinoid receptors and stress-responsive mitogen-activated protein kinases (MAPKs) in the cytotoxic process. J774-1 cells were treated with Δ 8 -THC (0–20 μM) for up to 6 h. As measured by the MTT and LDH assays, Δ 8 -THC induced cell death of J774-1 cells in a concentration- and/or exposure time-dependent manner. Δ 8 -THC-induced cell damage was associated with vacuole formation, cell swelling, chromatin condensation, and nuclear fragmentation. The cytotoxic effect of Δ 8 -THC was significantly prevented by a caspase-1 inhibitor Ac-YVAD-cmk but not a caspase-3 inhibitor z-DEVD-fmk. The pretreatment with SR144528, a CB 2 receptor-selective antagonist, effectively suppressed Δ 8 -THC-induced cytotoxicity in J774-1 cells, which exclusively expressed CB 2 receptors as indicated by real-time polymerase chain reaction analysis. In contrast, AM251, a CB 1 receptor-selective antagonist, did not affect the cytotoxicity. Pertussis toxin and α-tocopherol significantly attenuated Δ 8 -THC-induced cytotoxicity suggesting that G i/o protein coupling signal transduction and oxidative stress are responsible for the cytotoxicity. Δ 8 -THC stimulated the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) in J774-1 cells, which were effectively antagonized by the pretreatment with SR144528. In addition, SB203580, a p38 MARK inhibitor, significantly attenuated the cytotoxic effect of Δ 8 -THC, whereas SP600125, a JNK inhibitor, significantly enhanced the cytotoxicity. These results suggest that the cytotoxicity of Δ 8 -THC to J774-1 cells is exerted mediated through the CB 2 receptor followed by the activation of p38 MAPK

Novel Indole-based Tambjamine-Analogues Induce Apoptotic Lung Cancer Cell Death through p38 Mitogen-Activated Protein Kinase Activation.

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Manuel-Manresa, Pilar; Korrodi-Gregório, Luís; Hernando, Elsa; Villanueva, Alberto; Martínez-García, David; Rodilla, Ananda M; Ramos, Ricard; Fardilha, Margarida; Moya, Juan; Quesada, Roberto; Soto-Cerrato, Vanessa; Pérez-Tomás, Ricardo

2017-07-01

Lung cancer has become the leading killer cancer worldwide, due to late diagnosis and lack of efficient anticancer drugs. We have recently described novel natural-derived tambjamine analogues that are potent anion transporters capable of disrupting cellular ion balance, inducing acidification of the cytosol and hyperpolarization of cellular plasma membranes. Although these tambjamine analogues were able to compromise cell survival, their molecular mechanism of action remains largely unknown. Herein we characterize the molecular cell responses induced by highly active indole-based tambjamine analogues treatment in lung cancer cells. Expression changes produced after compounds treatment comprised genes related to apoptosis, cell cycle, growth factors and its receptors, protein kinases and topoisomerases, among others. Dysregulation of BCL2 and BIRC5 /survivin genes suggested the apoptotic pathway as the induced molecular cell death mechanism. In fact, activation of several proapoptotic markers (caspase-9, caspase-3, and PARP) and reversion of the cytotoxic effect upon treatment with an apoptosis inhibitor (Z-VAD-FMK) were observed. Moreover, members of the Bcl-2 protein family suffered changes after tambjamine analogues treatment, with a concomitant protein decrease towards the prosurvival members. Besides this, it was observed cellular accumulation of ROS upon compound treatment and an activation of the stress-kinase p38 MAPK route that, when inhibited, reverted the cytotoxic effect of the tambjamine analogues. Finally, a significant therapeutic effect of these compounds was observed in subcutaneous and orthotopic lung cancer mice models. Taken together, these results shed light on the mechanism of action of novel cytotoxic anionophores and demonstrate the therapeutic effects against lung cancer. Mol Cancer Ther; 16(7); 1224-35. ©2017 AACR . ©2017 American Association for Cancer Research.

PMK-1 p38 MAPK promotes cadmium stress resistance, the expression of SKN-1/Nrf and DAF-16 target genes, and protein biosynthesis in Caenorhabditis elegans.

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Keshet, Alex; Mertenskötter, Ansgar; Winter, Sarah A; Brinkmann, Vanessa; Dölling, Ramona; Paul, Rüdiger J

2017-12-01

The mechanisms of cadmium (Cd) resistance are complex and not sufficiently understood. The present study, therefore, aimed at assessing the roles of important components of stress-signaling pathways and of ABC transporters under severe Cd stress in Caenorhabditis elegans. Survival assays on mutant and control animals revealed a significant promotion of Cd resistance by the PMK-1 p38 MAP kinase, the transcription factor DAF-16/FoxO, and the ABC transporter MRP-1. Transcriptome profiling by RNA-Seq on wild type and a pmk-1 mutant under control and Cd stress conditions revealed, inter alia, a PMK-1-dependent promotion of gene expression for the translational machinery. PMK-1 also promoted the expression of target genes of the transcription factors SKN-1/Nrf and DAF-16 in Cd-stressed animals, which included genes for molecular chaperones or immune proteins. Gene expression studies by qRT-PCR confirmed the positive effects of PMK-1 on DAF-16 activity under Cd stress and revealed negative effects of DAF-16 on the expression of genes for MRP-1 and DAF-15/raptor. Additional studies on pmk-1 RNAi-treated wild type and mutant strains provided further information on the effects of PMK-1 on SKN-1 and DAF-16, which resulted in a model of these relationships. The results of this study demonstrate a central role of PMK-1 for the processing of cellular responses to abiotic and biotic stressors, with the promoting effects of PMK-1 on Cd resistance mostly mediated by the transcription factors SKN-1 and DAF-16.

Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

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Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

2003-09-18

The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

Chrysin protects against cisplatin-induced colon. toxicity via amelioration of oxidative stress and apoptosis: Probable role of p38MAPK and p53

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Khan, Rehan; Khan, Abdul Quaiyoom; Qamar, Wajhul; Lateef, Abdul; Tahir, Mir; Rehman, Muneeb U; Ali, Farrah; Sultana, Sarwat, E-mail: sarwat786@rediffmail.com

2012-02-01

Cisplatin, an antineoplastic drug, is widely used as a foremost therapy against numerous forms of cancer but it has pronounced adverse effects viz., nephrotoxicity, ototoxicity etc. CDDP-induced emesis and diarrhea are also marked toxicities that may be due to intestinal injury. Chrysin (5,7-dihydroxyflavone), a natural flavone commonly found in many plants possesses multiple biological activities, such as antioxidant, anti-inflammatory and anti-cancer effects. In the present study, we investigated the protective effect of chrysin against CDDP-induced colon toxicity. The plausible mechanism of CDDP-induced colon toxicity and damage includes oxidative stress, activation of p38MAPK and p53, and colonic epithelial cell apoptosis via upregulating the expression of Bak and cleaved caspase-3. Chrysin was administered to Wistar rats once daily for 14 consecutive days at the doses of 25 and 50 mg/kg body weight orally in corn oil. On day 14, a single intraperitoneal injection of cisplatin was given at the dose of 7.5 mg/kg body weight and animals were euthanized after 24 h of cisplatin injection. Chrysin ameliorated CDDP-induced lipid peroxidation, xanthine oxidase activity, glutathione depletion, decrease in antioxidant (catalase, glutathione reductase, glutathione peroxidase and glucose-6 phosphate dehydrogenase) and phase-II detoxifying (glutathione-S-transferase and quinone reductase) enzyme activities. Chrysin also attenuated goblet cell disintegration, expression of phospho-p38MAPK and p53, and apoptotic tissue damage which were induced by CDDP. Histological findings further supported the protective effects of chrysin against CDDP-induced colonic damage. The results of the present study suggest that the protective effect of chrysin against CDDP-induced colon toxicity was related with attenuation of oxidative stress, activation of p38MAPK and p53, and apoptotic tissue damage. Highlights: ► Cisplatin-induced colon toxicity is associated with oxidative stress and

Chrysin protects against cisplatin-induced colon. toxicity via amelioration of oxidative stress and apoptosis: Probable role of p38MAPK and p53

International Nuclear Information System (INIS)

Khan, Rehan; Khan, Abdul Quaiyoom; Qamar, Wajhul; Lateef, Abdul; Tahir, Mir; Rehman, Muneeb U; Ali, Farrah; Sultana, Sarwat

2012-01-01

Cisplatin, an antineoplastic drug, is widely used as a foremost therapy against numerous forms of cancer but it has pronounced adverse effects viz., nephrotoxicity, ototoxicity etc. CDDP-induced emesis and diarrhea are also marked toxicities that may be due to intestinal injury. Chrysin (5,7-dihydroxyflavone), a natural flavone commonly found in many plants possesses multiple biological activities, such as antioxidant, anti-inflammatory and anti-cancer effects. In the present study, we investigated the protective effect of chrysin against CDDP-induced colon toxicity. The plausible mechanism of CDDP-induced colon toxicity and damage includes oxidative stress, activation of p38MAPK and p53, and colonic epithelial cell apoptosis via upregulating the expression of Bak and cleaved caspase-3. Chrysin was administered to Wistar rats once daily for 14 consecutive days at the doses of 25 and 50 mg/kg body weight orally in corn oil. On day 14, a single intraperitoneal injection of cisplatin was given at the dose of 7.5 mg/kg body weight and animals were euthanized after 24 h of cisplatin injection. Chrysin ameliorated CDDP-induced lipid peroxidation, xanthine oxidase activity, glutathione depletion, decrease in antioxidant (catalase, glutathione reductase, glutathione peroxidase and glucose-6 phosphate dehydrogenase) and phase-II detoxifying (glutathione-S-transferase and quinone reductase) enzyme activities. Chrysin also attenuated goblet cell disintegration, expression of phospho-p38MAPK and p53, and apoptotic tissue damage which were induced by CDDP. Histological findings further supported the protective effects of chrysin against CDDP-induced colonic damage. The results of the present study suggest that the protective effect of chrysin against CDDP-induced colon toxicity was related with attenuation of oxidative stress, activation of p38MAPK and p53, and apoptotic tissue damage. Highlights: ► Cisplatin-induced colon toxicity is associated with oxidative stress and

Microwave-assisted synthesis of 5-aminopyrazol-4-yl ketones and the p38(MAPK) inhibitor RO3201195 for study in Werner syndrome cells.

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Bagley, Mark C; Davis, Terence; Dix, Matthew C; Murziani, Paola G S; Rokicki, Michal J; Kipling, David

2008-07-01

5-Aminopyrazol-4-yl ketones are prepared rapidly and efficiently using microwave dielectric heating from beta-ketonitriles by treatment with N,N'-diphenylformamidine followed by heterocyclocondensation by irradiation with a hydrazine. The inhibitory activity of RO3201195 prepared by this methodology was confirmed in hTERT-immortalized HCA2 and WS dermal fibroblasts at 200nM concentration, both by ELISA and immunoblot assay, and displays excellent kinase selectivity for p38alpha MAPK over the related stress-activated kinase JNK.

Effect of 3G cell phone exposure with computer controlled 2-D stepper motor on non-thermal activation of the hsp27/p38MAPK stress pathway in rat brain.

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Kesari, Kavindra Kumar; Meena, Ramovatar; Nirala, Jayprakash; Kumar, Jitender; Verma, H N

2014-03-01

Cell phone radiation exposure and its biological interaction is the present concern of debate. Present study aimed to investigate the effect of 3G cell phone exposure with computer controlled 2-D stepper motor on 45-day-old male Wistar rat brain. Animals were exposed for 2 h a day for 60 days by using mobile phone with angular movement up to zero to 30°. The variation of the motor is restricted to 90° with respect to the horizontal plane, moving at a pre-determined rate of 2° per minute. Immediately after 60 days of exposure, animals were scarified and numbers of parameters (DNA double-strand break, micronuclei, caspase 3, apoptosis, DNA fragmentation, expression of stress-responsive genes) were performed. Result shows that microwave radiation emitted from 3G mobile phone significantly induced DNA strand breaks in brain. Meanwhile a significant increase in micronuclei, caspase 3 and apoptosis were also observed in exposed group (P 3G mobile phone exposure causes a transient increase in phosphorylation of hsp27, hsp70, and p38 mitogen-activated protein kinase (p38MAPK), which leads to mitochondrial dysfunction-mediated cytochrome c release and subsequent activation of caspases, involved in the process of radiation-induced apoptotic cell death. Study shows that the oxidative stress is the main factor which activates a variety of cellular signal transduction pathways, among them the hsp27/p38MAPK is the pathway of principle stress response. Results conclude that 3G mobile phone radiations affect the brain function and cause several neurological disorders.

Differential activation of p38 and extracellular signal-regulated kinase in spinal cord in a model of bee venom-induced inflammation and hyperalgesia

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Kobayashi Kimiko

2008-04-01

Full Text Available Abstract Background Honeybee's sting on human skin can induce ongoing pain, hyperalgesia and inflammation. Injection of bee venom (BV into the intraplantar surface of the rat hindpaw induces an early onset of spontaneous pain followed by a lasting thermal and mechanical hypersensitivity in the affected paw. The underlying mechanisms of BV-induced thermal and mechanical hypersensitivity are, however, poorly understood. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK in the generation of BV-induced pain hypersensitivity. Results We found that BV injection resulted in a quick activation of p38, predominantly in the L4/L5 spinal dorsal horn ipsilateral to the inflammation from 1 hr to 7 d post-injection. Phosphorylated p38 (p-p38 was expressed in both neurons and microglia, but not in astrocytes. Intrathecal administration of the p38 inhibitor, SB203580, prevented BV-induced thermal hypersensitivity from 1 hr to 3 d, but had no effect on mechanical hypersensitivity. Activated ERK1/2 was observed exclusively in neurons in the L4/L5 dorsal horn from 2 min to 1 d, peaking at 2 min after BV injection. Intrathecal administration of the MEK inhibitor, U0126, prevented both mechanical and thermal hypersensitivity from 1 hr to 2 d. p-ERK1/2 and p-p38 were expressed in neurons in distinct regions of the L4/L5 dorsal horn; p-ERK1/2 was mainly in lamina I, while p-p38 was mainly in lamina II of the dorsal horn. Conclusion The results indicate that differential activation of p38 and ERK1/2 in the dorsal horn may contribute to the generation and development of BV-induced pain hypersensitivity by different mechanisms.

Geraniol attenuates 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammation in mouse skin: possible role of p38 MAP Kinase and NF-κB.

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Khan, Abdul Quaiyoom; Khan, Rehan; Qamar, Wajhul; Lateef, Abdul; Rehman, Muneeb U; Tahir, Mir; Ali, Farrah; Hamiza, Oday O; Hasan, Syed Kazim; Sultana, Sarwat

2013-06-01

Abnormal production of reactive oxygen species (ROS) and proinflammatory cytokines often act as trigger for development of most of the chronic human diseases including cancer via up-regulation of transcription factors and activation of MAP kinases. We investigated the protective effects of geraniol (GOH) against 12-O-tetradecanoyl phorbol-13-acetate (TPA) induced oxidative and inflammatory responses, expression of p38MAPK, NF-κB and COX-2 in mouse skin. Animals were divided into four groups I-IV (n=6). Group II and III received topical application of TPA at the dose of 10 nmol/0.2 ml of acetone/animal/day, for two days. Group III was pre-treated with GOH (250 μg) topically 30 min prior to each TPA administration. While group I and IV were given acetone (0.2 ml) and GOH respectively. Our results show that GOH significantly inhibited TPA induced lipid peroxidation (LPO), inflammatory responses, proinflammatory cytokine release, up regulates reduced glutathione (GSH) content and the activity of different antioxidant enzymes. Interestingly, GOH also inhibited TPA induced altered activity of p38MAPK. Further, TPA induced altered expression of NF-κB (p65) and COX-2 was also attenuated by GOH. Thus, our results suggest that GOH attenuates early tumor promotional changes, and it may serve as one of the various ways to prevent carcinogenesis. Copyright © 2013 Elsevier Inc. All rights reserved.

TGF-β1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38 MAPK.

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Witte, David; Bartscht, Tobias; Kaufmann, Roland; Pries, Ralph; Settmacher, Utz; Lehnert, Hendrik; Ungefroren, Hendrik

2017-12-01

Transforming growth factor (TGF)-β promotes epithelial-mesenchymal transition and cell invasion of cancer cells in part through the small GTPase RAC1. Since RAC1 can signal through reactive oxygen species (ROS), we probed the role of the ROS-producing NADPH oxidase (NOX) and p38 mitogen-activated protein kinase (MAPK) in mediating TGF-β1/RAC1-driven random cell migration (chemokinesis). Although the NOX isoforms NOX2, 4, 5, 6, and RAC1 were readily detectable by RT-PCR in pancreatic ductal adenocarcinoma (PDAC)-derived Panc1 and Colo357 cells, only NOX4 and RAC1 were expressed at higher levels comparable to those in peripheral blood monocytes. TGF-β1 treatment resulted in upregulation of NOX4 (and NOX2) and rapid intracellular production of ROS. To analyze whether RAC1 functions through NOX and ROS to promote cell motility, we performed real-time cell migration assays with xCELLigence® technology in the presence of the ROS scavenger N-acetyl-L-cysteine (NAC) and various NOX inhibitors. NAC, the NOX4 inhibitor diphenylene iodonium or small interfering RNA (siRNA) to NOX4, and the NOX2 inhibitor apocynin all suppressed TGF-β1-induced chemokinesis of Panc1 and Colo357 cells as did various inhibitors of RAC1 used as control. In addition, we showed that blocking NOX4 or RAC1 function abrogated phosphorylation of p38 MAPK signaling by TGF-β1 and that inhibition of p38 MAPK reduced TGF-β1-induced random cell migration, while ectopic expression of a kinase-active version of the p38 activating kinase MKK6 was able to partially rescue the decline in migration after RAC1 inhibition. Our data suggest that TGF-β1-induced chemokinesis in PDAC cells is mediated through a RAC1/NOX4/ROS/p38 MAPK cascade.

Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus

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Kyle K. Biggar

2015-04-01

Full Text Available Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK family members to primate torpor were compared in six organs of control (aroused versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs, c-jun NH2-terminal kinases (JNKs, MAPK kinase (MEK, and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27. The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor.

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

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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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

p38 MAP kinase is required for Wnt3a-mediated osterix expression independently of Wnt-LRP5/6-GSK3β signaling axis in dental follicle cells

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Sakisaka, Yukihiko; Kanaya, Sousuke; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi; Nemoto, Eiji

2016-01-01

Wnt3a is a secreted glycoprotein that activates the glycogen synthase kinase-3β (GSK3β)/β-catenin signaling pathway through low-density-lipoprotein receptor-related protein (LRP)5/6 co-receptors. Wnt3a has been implicated in periodontal development and homeostasis, as well as in cementum formation. Recently, we have reported that Wnt3a increases alkaline phosphatase expression through the induction of osterix (Osx) expression in dental follicle cells, a precursor of cementoblasts. However, the molecular mechanism by which Wnt3a induces Osx expression is still unknown. In this study, we show that Wnt3a-induced Osx expression was inhibited in the presence of p38 mitogen-activated protein kinase (MAPK) inhibitors (SB203580 and SB202190) at gene and protein levels, as assessed by real-time PCR and immunocytohistochemistry, respectively. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist binding to LRP5/6 co-receptors, did not influence Wnt3a-mediated p38 MAPK phosphorylation, suggesting that Wnt3a activates p38 MAPK through LRP5/6-independent signaling. On the other hand, pretreatment with p38 MAPK inhibitors had no effects on the phosphorylated status of GSK3β and β-catenin as well as β-catenin nuclear translocation, but inhibited Wnt3a-mediated β-catenin transcriptional activity. These findings suggest that p38 MAPK modulates canonical Wnt signaling at the β-catenin transcriptional level without any crosstalk with the Wnt3a-mediated LRP5/6-GSK3β signaling axis and subsequent β-catenin nuclear translocation. These findings expand our knowledge of the mechanisms controlling periodontal development and regeneration. - Highlights: • Wnt3a induces Osx expression via p38 MAPK signaling in dental follicle cells. • p38 MAPK has no crosstalk with Wnt3a-mediated LRP5/6 and GSK3β signaling. • p38 MAPK is required for Wnt signaling at the β-catenin transcriptional level.

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

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

2002-05-01

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

Characterization of p38 MAPK isoforms for drug resistance study using systems biology approach.

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Peng, Huiming; Peng, Tao; Wen, Jianguo; Engler, David A; Matsunami, Risë K; Su, Jing; Zhang, Le; Chang, Chung-Che Jeff; Zhou, Xiaobo

2014-07-01

p38 mitogen-activated protein kinase activation plays an important role in resistance to chemotherapeutic cytotoxic drugs in treating multiple myeloma (MM). However, how the p38 mitogen-activated protein kinase signaling pathway is involved in drug resistance, in particular the roles that the various p38 isoforms play, remains largely unknown. To explore the underlying mechanisms, we developed a novel systems biology approach by integrating liquid chromatography-mass spectrometry and reverse phase protein array data from human MM cell lines with computational pathway models in which the unknown parameters were inferred using a proposed novel algorithm called modularized factor graph. New mechanisms predicted by our models suggest that combined activation of various p38 isoforms may result in drug resistance in MM via regulating the related pathways including extracellular signal-regulated kinase (ERK) pathway and NFкB pathway. ERK pathway regulating cell growth is synergistically regulated by p38δ isoform, whereas nuclear factor kappa B (NFкB) pathway regulating cell apoptosis is synergistically regulated by p38α isoform. This finding that p38δ isoform promotes the phosphorylation of ERK1/2 in MM cells treated with bortezomib was validated by western blotting. Based on the predicted mechanisms, we further screened drug combinations in silico and found that a promising drug combination targeting ERK1/2 and NFκB might reduce the effects of drug resistance in MM cells. This study provides a framework of a systems biology approach to studying drug resistance and drug combination selection. RPPA experimental Data and Matlab source codes of modularized factor graph for parameter estimation are freely available online at http://ctsb.is.wfubmc.edu/publications/modularized-factor-graph.php. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A Member of the p38 Mitogen-Activated Protein Kinase Family Is Responsible for Transcriptional Induction of Dopa decarboxylase in the Epidermis of Drosophila melanogaster during the Innate Immune Response▿ †

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Davis, Monica M.; Primrose, David A.; Hodgetts, Ross B.

2008-01-01

Drosophila innate immunity is controlled primarily by the activation of IMD (immune deficiency) or Toll signaling leading to the production of antimicrobial peptides (AMPs). IMD signaling also activates the JUN N-terminal kinase (JNK) cascade, which is responsible for immune induction of non-antimicrobial peptide immune gene transcription though the transcription factor AP-1. Transcription of the Dopa decarboxylase (Ddc) gene is induced in response to gram-negative and gram-positive septic injury, but not aseptic wounding. Transcription is induced throughout the epidermis and not specifically at the site of infection. Ddc transcripts are detectible within 2 h and remain high for several hours following infection with either gram-negative or gram-positive bacteria. Using Ddc-green fluorescent protein (GFP) reporter gene constructs, we show that a conserved consensus AP-1 binding site upstream of the Ddc transcription start site is required for induction. However, neither the Toll, IMD, nor JNK pathway is involved. Rather, Ddc transcription depends on a previously uncharacterized member of the p38 mitogen-activated protein kinase family, p38c. We propose that the involvement of DDC in a new pathway involved in Drosophila immunity increases the levels of dopamine, which is metabolized to produce reactive quinones that exert an antimicrobial effect on invading bacteria. PMID:18519585

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

DEFF Research Database (Denmark)

Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

2013-01-01

knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

Regulation of Autophagy by Kinases

International Nuclear Information System (INIS)

Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

2011-01-01

Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

Regulation of Autophagy by Kinases

Energy Technology Data Exchange (ETDEWEB)

Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

2011-06-09

Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

Regulation of Autophagy by Kinases

Science.gov (United States)

Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

2011-01-01

Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets. PMID:24212825

Regulation of Autophagy by Kinases

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Savitha Sridharan

2011-06-01

Full Text Available Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

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

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

2016-12-09

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

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

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Kang-Hyun Leem

2016-12-01

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

Gram-scale synthesis of the p38α MAPK-inhibitor VX-745 for preclinical studies into Werner syndrome.

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Bagley, Mark C; Davis, Terence; Dix, Matthew C; Fusillo, Vincenzo; Pigeaux, Morgane; Rokicki, Michal J; Kipling, David

2010-09-01

The ATP-competitive p38α MAPK inhibitor VX-745 exhibits an exquisite kinase selectivity profile, is effective in blocking p38 stress signaling in Werner syndrome dermal fibroblasts, has efficacy in clinical trials and may have therapeutic value against Werner syndrome. Previous synthetic routes, however, have only resulted in milligram quantities suitable for cell-based studies, whereas gram quantities would be required for in vivo use. Microwave irradiation using a stop-flow monomodal microwave reactor has been found to facilitate scale-up of the synthesis of VX-745. Ullmann-type C-S bond formation using thiophenol, chloropyridazine, copper(I) catalyst and diol ligand proceeds rapidly and efficiently in this apparatus for elaboration to the pyrimido[1,6-b]pyridazinone core of VX-745 on gram scale and with good overall yield. This method delivers the p38 inhibitor VX-745 in sufficient quantities for preclinical studies to rescue the aging phenotype in Werner syndrome.

Redox-sensitive up-regulation of eNOS by purple grape juice in endothelial cells: role of PI3-kinase/Akt, p38 MAPK, JNK, FoxO1 and FoxO3a.

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Mahmoud Alhosin

Full Text Available The vascular protective effect of grape-derived polyphenols has been attributable, in part, to their direct action on blood vessels by stimulating the endothelial formation of nitric oxide (NO. The aim of the present study was to determine whether Concord grape juice (CGJ, which contains high levels of polyphenols, stimulates the expression of endothelial NO synthase (eNOS in porcine coronary artery endothelial cells and, if so, to determine the signaling pathway involved. CGJ dose- and time-dependently increased eNOS mRNA and protein levels and this effect is associated with an increased formation of NO in endothelial cells. The stimulatory effect of CGJ on eNOS mRNA is not associated with an increased eNOS mRNA stability and inhibited by antioxidants such as MnTMPyP, PEG-catalase, and catalase, and by wortmannin (an inhibitor of PI3-kinase, SB 203580 (an inhibitor of p38 MAPK, and SP 600125 (an inhibitor of JNK. Moreover, CGJ induced the formation of reactive oxygen species (ROS in endothelial cells and this effect is inhibited by MnTMPyP, PEG-catalase, and catalase. The CGJ-induced the phosphorylation of p38 MAPK and JNK kinases is abolished by MnTMPyP. CGJ induced phosphorylation of transcription factors FoxO1 and FoxO3a, which regulate negatively eNOS expression, and this effect is prevented by MnTMPyP, PEG-catalase, wortmannin, SB203580 and SP600125. Moreover, chromatin immunoprecipitation assay indicated that the FoxO3a protein is associated with the eNOS promoter in control cells and that CGJ induced its dissociation. Thus, the present study indicates that CGJ up-regulates the expression of eNOS mRNA and protein leading to an increased formation of NO in endothelial cells. The stimulatory effect of CGJ is a redox-sensitive event involving PI3-kinase/Akt, p38 MAPK and JNK pathways, and the inactivation of the FoxO transcription factors, FoxO1 and FoxO3a, thereby preventing their repression of the eNOS gene.

Arabidopsis calcium-dependent protein kinase AtCPK1 plays a positive role in salt/drought-stress response.

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Huang, Kui; Peng, Lu; Liu, Yingying; Yao, Rundong; Liu, Zhibin; Li, Xufeng; Yang, Yi; Wang, Jianmei

2018-03-25

The calcium-dependent protein kinases (CDPKs) play vital roles in plant response to various environmental stimuli. Here, we investigated the function of Arabidopsis AtCPK1 in response to salt and drought stress. The loss-of-function cpk1 mutant displayed hypersensitive to salt and drought stress, whereas overexpressing AtCPK1 in Arabidopsis plants significantly enhanced the resistance to salt or drought stress. The reduced or elevated tolerance of cpk1 mutant and AtCPK1-overexpressing lines was confirmed by the changes of proline, malondialdehyde (MDA) and H 2 O 2 . Real-time PCR analysis revealed that the expression of several stress-inducible genes (RD29A, COR15A, ZAT10, APX2) down-regulated in cpk1 mutant and up-regulated in AtCPK1-overexpressing plants. These results are likely to indicate that AtCPK1 positively regulates salt and drought stress in Arabidopsis. Copyright © 2017 Elsevier Inc. All rights reserved.

Seasonal variations of cellular stress response in the heart and gastrocnemius muscle of the water frog (Pelophylax ridibundus).

Science.gov (United States)

Feidantsis, Konstantinos; Anestis, Andreas; Vasara, Eleni; Kyriakopoulou-Sklavounou, Pasqualina; Michaelidis, Basile

2012-08-01

The present study aimed to investigate the seasonal cellular stress response in the heart and the gastrocnemius muscle of the amphibian Pelophylax ridibundus (former name Rana ridibunda) during an 8 month acclimatization period in the field. Processes studied included heat shock protein expression and protein kinase activation. The cellular stress response was addressed through the expression of Hsp70 and Hsp90 and the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK), the extracellular signal-regulated kinases (ERK-1/2) and c-Jun N-terminal kinases (JNK1/2/3). Due to a general metabolic depression during winter hibernation, the induction of Hsp70 and Hsp90 and the phosphorylation of p38 MAPK, JNKs and ERKs are retained at low levels of expression in the examined tissues of P. ridibundus. Recovery from hibernation induces increased levels of the specific proteins, probably providing stamina to the animals during their arousal. Copyright © 2012 Elsevier Inc. All rights reserved.

A systematic review of p53 regulation of oxidative stress in skeletal muscle.

Science.gov (United States)

Beyfuss, Kaitlyn; Hood, David A

2018-12-01

p53 is a tumor suppressor protein involved in regulating a wide array of signaling pathways. The role of p53 in the cell is determined by the type of imposed oxidative stress, its intensity and duration. The last decade of research has unravelled a dual nature in the function of p53 in mediating the oxidative stress burden. However, this is dependent on the specific properties of the applied stress and thus requires further analysis. A systematic review was performed following an electronic search of Pubmed, Google Scholar, and ScienceDirect databases. Articles published in the English language between January 1, 1990 and March 1, 2017 were identified and isolated based on the analysis of p53 in skeletal muscle in both animal and cell culture models. Literature was categorized according to the modality of imposed oxidative stress including exercise, diet modification, exogenous oxidizing agents, tissue manipulation, irradiation, and hypoxia. With low to moderate levels of oxidative stress, p53 is involved in activating pathways that increase time for cell repair, such as cell cycle arrest and autophagy, to enhance cell survival. However, with greater levels of stress intensity and duration, such as with irradiation, hypoxia, and oxidizing agents, the role of p53 switches to facilitate increased cellular stress levels by initiating DNA fragmentation to induce apoptosis, thereby preventing aberrant cell proliferation. Current evidence confirms that p53 acts as a threshold regulator of cellular homeostasis. Therefore, within each modality, the intensity and duration are parameters of the oxidative stressor that must be analyzed to determine the role p53 plays in regulating signaling pathways to maintain cellular health and function in skeletal muscle. Acadl: acyl-CoA dehydrogenase, long chain; Acadm: acyl-CoA dehydrogenase, C-4 to C-12 straight chain; AIF: apoptosis-inducing factor; Akt: protein kinase B (PKB); AMPK: AMP-activated protein kinase; ATF-4: activating

p38 MAPK Inhibitor Insufficiently Attenuates HSC Senescence Administered Long-Term after 6 Gy Total Body Irradiation in Mice

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Lu Lu

2016-06-01

Full Text Available Senescent hematopoietic stem cells (HSCs accumulate with age and exposure to stress, such as total-body irradiation (TBI, which may cause long-term myelosuppression in the clinic. However, the methods available for long-term myelosuppression remain limited. Previous studies have demonstrated that sustained p38 mitogen-activated protein kinases (p38 MAPK activation in HSCs following exposure to TBI in mice and the administration of its inhibitor twenty-four hours after TBI may partially prevent long-term myelosuppression. However, long-term myelosuppression is latent and identified long after the administration of radiation. In this study, we investigated the effects of SB203580 (a small molecule inhibitor of p38 MAPK on long-term myelosuppression induced by TBI. Mice with hematopoietic injury were injected intraperitoneally with SB203580 every other day five times beginning 70 days after 6 Gy of 137Cs γ ray TBI. Our results at 80 days demonstrated that SB203580 did not significantly improve the TBI-induced long-term reduction of peripheral blood cell and bone marrow nucleated cell (BMNC counts, or defects in hematopoietic progenitor cells (HPCs and HSC clonogenic function. SB203580 reduced reactive oxygen species (ROS production and p-p38 expression; however, SB203580 had no effect on p16 expression in the HSCs of mice. In conclusion, these findings suggest that treatment with SB203580 70 days after TBI in mice inhibits the ROS-p38 oxidative stress pathway; however, it has no therapeutic effect on long-term myelosuppression induced by TBI.

Lysophosphatidylcholine Promotes Phagosome Maturation and Regulates Inflammatory Mediator Production Through the Protein Kinase A–Phosphatidylinositol 3 Kinase–p38 Mitogen-Activated Protein Kinase Signaling Pathway During Mycobacterium tuberculosis Infection in Mouse Macrophages

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Hyo-Ji Lee

2018-04-01

Full Text Available Tuberculosis is caused by the infectious agent Mycobacterium tuberculosis (Mtb. Mtb has various survival strategies, including blockade of phagosome maturation and inhibition of antigen presentation. Lysophosphatidylcholine (LPC is a major phospholipid component of oxidized low-density lipoprotein and is involved in various cellular responses, such as activation of second messengers and bactericidal activity in neutrophils. In this study, macrophages were infected with a low infectious dose of Mtb and treated with LPC to investigate the bactericidal activity of LPC against Mtb. In macrophages infected with Mtb strain, H37Ra or H37Rv, LPC suppressed bacterial growth; however, this effect was suppressed in bone marrow-derived macrophages (BMDMs isolated from G2A (a G protein-coupled receptor involved in some LPC actions knockout mice. LPC also promoted phagosome maturation via phosphatidylinositol 3 kinase (PI3K–p38 mitogen-activated protein kinase (MAPK-mediated reactive oxygen species production and intracellular Ca2+ release during Mtb infection. In addition, LPC induced increased levels of intracellular cyclic adenosine monophosphate (cAMP and phosphorylated glycogen synthase kinase 3 beta (GSK3β in Mtb-infected macrophages. Protein kinase A (PKA-induced phosphorylation of GSK3β suppressed activation of NF-κB in LPC-treated macrophages during Mtb infection, leading to decreased secretion of pro-inflammatory cytokines and increased secretion of anti-inflammatory cytokines. These results suggest that LPC can effectively control Mtb growth by promoting phagosome maturation via cAMP-induced activation of the PKA–PI3K–p38 MAPK pathway. Moreover, LPC can regulate excessive production of pro-inflammatory cytokines associated with bacterial infection of macrophages.

Desmoglein 2 regulates the intestinal epithelial barrier via p38 mitogen-activated protein kinase.

Science.gov (United States)

Ungewiß, Hanna; Vielmuth, Franziska; Suzuki, Shintaro T; Maiser, Andreas; Harz, Hartmann; Leonhardt, Heinrich; Kugelmann, Daniela; Schlegel, Nicolas; Waschke, Jens

2017-07-24

Intestinal epithelial barrier properties are maintained by a junctional complex consisting of tight junctions (TJ), adherens junctions (AJ) and desmosomes. Desmoglein 2 (Dsg2), an adhesion molecule of desmosomes and the only Dsg isoform expressed in enterocytes, is required for epithelial barrier properties and may contribute to barrier defects in Crohn's disease. Here, we identified extradesmosomal Dsg2 on the surface of polarized enterocytes by Triton extraction, confocal microscopy, SIM and STED. Atomic force microscopy (AFM) revealed Dsg2-specific binding events along the cell border on the surface of enterocytes with a mean unbinding force of around 30pN. Binding events were blocked by an inhibitory antibody targeting Dsg2 which under same conditions activated p38MAPK but did not reduce cell cohesion. In enterocytes deficient for Dsg2, p38MAPK activity was reduced and both barrier integrity and reformation were impaired. Dsc2 rescue did not restore p38MAPK activity indicating that Dsg2 is required. Accordingly, direct activation of p38MAPK in Dsg2-deficient cells enhanced barrier reformation demonstrating that Dsg2-mediated activation of p38MAPK is crucial for barrier function. Collectively, our data show that Dsg2, beside its adhesion function, regulates intestinal barrier function via p38MAPK signalling. This is in contrast to keratinocytes and points towards tissue-specific signalling functions of desmosomal cadherins.

The p38 mitogen activated protein kinase regulates β-amyloid protein internalization through the α7 nicotinic acetylcholine receptor in mouse brain.

Science.gov (United States)

Ma, Kai-Ge; Lv, Jia; Yang, Wei-Na; Chang, Ke-Wei; Hu, Xiao-Dan; Shi, Li-Li; Zhai, Wan-Ying; Zong, Hang-Fan; Qian, Yi-Hua

2018-03-01

Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders. Intracellular β-amyloid protein (Aβ) is an early event in AD. It induces the formation of amyloid plaques and neuron damage. The α7 nicotinic acetylcholine receptor (α7nAChR) has been suggested to play an important role in Aβ caused cognition. It has high affinity with Aβ and could mediate Aβ internalization in vitro. However, whether in mouse brain the p38 MAPK signaling pathway is involved in the regulation of the α7nAChR mediated Aβ internalization and their role in mitochondria remains little known. Therefore, in this study, we revealed that Aβ is internalized by cholinergic and GABAergic neurons. The internalized Aβ were found deposits in lysosomes/endosomes and mitochondria. Aβ could form Aβ-α7nAChR complex with α7nAChR, activates the p38 mitogen activated protein kinase (MAPK). And the increasing of α7nAChR could in return mediate Aβ internalization in the cortex and hippocampus. In addition, by using the α7nAChR agonist PNU282987, the p38 phosphorylation level decreases, rescues the biochemical changes which are tightly associated with Aβ-induced apoptosis, such as Bcl2/Bax level, cytochrome c (Cyt c) release. Collectively, the p38 MAPK signaling pathway could regulate the α7nAChR-mediated internalization of Aβ. The activation of α7nAChR or the inhibition of p38 MAPK signaling pathway may be a beneficial therapy to AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Phosphorylation of Icariin Can Alleviate the Oxidative Stress Caused by the Duck Hepatitis Virus A through Mitogen-Activated Protein Kinases Signaling Pathways

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Wen Xiong

2017-09-01

Full Text Available The duck virus hepatitis (DVH caused by the duck hepatitis virus A (DHAV has produced extensive economic losses to the duck industry. The currently licensed commercial vaccine has shown some defects and does not completely prevent the DVH. Accordingly, a new alternative treatment for this disease is urgently needed. Previous studies have shown that icariin (ICA and its phosphorylated derivative (pICA possessed good anti-DHAV effects through direct and indirect antiviral pathways, such as antioxidative stress. But the antioxidant activity showed some differences between ICA and pICA. The aim of this study is to prove that ICA and pICA attenuate oxidative stress caused by DHAV in vitro and in vivo, and to investigate their mechanism of action to explain their differences in antioxidant activities. In vivo, the dynamic deaths, oxidative evaluation indexes and hepatic pathological change scores were detected. When was added the hinokitiol which showed the pro-oxidative effect as an intervention method, pICA still possessed more treatment effect than ICA. The strong correlation between mortality and oxidative stress proves that ICA and pICA alleviate oxidative stress caused by DHAV. This was also demonstrated by the addition of hydrogen peroxide (H2O2 as an intervention method in vitro. pICA can be more effective than ICA to improve duck embryonic hepatocytes (DEHs viability and reduce the virulence of DHAV. The strong correlation between TCID50 and oxidative stress demonstrates that ICA and pICA can achieve anti-DHAV effects by inhibiting oxidative stress. In addition, the superoxide dismutase (SOD and glutathione peroxidase (GSH-Px of ICA and pICA showed significant difference. pICA could significantly inhibit the phosphorylation of p38, extra cellular signal regulated Kinase (ERK 1/2 and c-Jun N-terminal kinase (JNK, which were related to mitogen-activated protein kinases (MAPKs signaling pathways. Ultimately, compared to ICA, pICA exhibited more

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

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Inger Lindin

2014-03-01

Full Text Available The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD simulations of: (1 MK5 alone; (2 MK5 in complex with an inhibitor; and (3 MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

Science.gov (United States)

Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

2011-11-01

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

Functional p53 in cells contributes to the anticancer effect of the cyclin-dependent kinase inhibitor roscovitine

Czech Academy of Sciences Publication Activity Database

Paprskářová, Martina; Kryštof, Vladimír; Jorda, Radek; Džubák, P.; Hajdúch, M.; Wesierska-Gadek, J.; Strnad, Miroslav

2009-01-01

Roč. 107, č. 3 (2009), s. 428-437 ISSN 0730-2312 R&D Projects: GA ČR GA204/08/0511 Institutional research plan: CEZ:AV0Z50380511 Keywords : APOPTOSIS * CYCLIN-DEPENDENT KINASE * OLOMOUCINE II * p53 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.935, year: 2009

Glycogen synthase kinase-3beta and the p25 activator of cyclin dependent kinase 5 increase pausing of mitochondria in neurons.

Science.gov (United States)

Morel, M; Authelet, M; Dedecker, R; Brion, J P

2010-06-02

The complex bi-directional axoplasmic transport of mitochondria is essential for proper metabolic functioning of neurons and is controlled by phosphorylation. We have investigated by time-lapse imaging the effects of increased expression of glycogen synthase kinase-3beta (GSK-3beta) and of the p25 activator of cyclin dependent kinase 5 on mitochondria movements in mammalian cortical neurons and in PC12 cells. Both GSK-3beta and p25 increased the stationary behaviour of mitochondria in PC12 and in neurons, decreased their anterograde transport but did not affect the intrinsic velocities of mitochondria. The microtubule-associated tau proteins were more phosphorylated in GSK-3beta and p25 transfected neurons, but ultrastructural observation showed that these cells still contained microtubules and nocodazole treatment further reduced residual mitochondria movements in GSK-3beta or p25 transfected neurons, indicating that microtubule disruption was not the primary cause of increased mitochondrial stationary behaviour in GSK-3beta or p25 transfected neurons. Our results suggest that increased expression of GSK-3beta and p25 acted rather by decreasing the frequency of mitochondrial movements driven by molecular motors and that GSK-3beta and p25 might regulate these transports by controlling the time that mitochondria spend pausing, rather than their velocities. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

P38 delta MAPK promotes breast cancer progression and lung metastasis by enhancing cell proliferation and cell detachment.

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Wada, M; Canals, D; Adada, M; Coant, N; Salama, M F; Helke, K L; Arthur, J S; Shroyer, K R; Kitatani, K; Obeid, L M; Hannun, Y A

2017-11-23

The protein p38 mitogen-activated protein kinase (MAPK) delta isoform (p38δ) is a poorly studied member of the MAPK family. Data analysis from The Cancer Genome Atlas database revealed that p38δ is highly expressed in all types of human breast cancers. Using a human breast cancer tissue array, we confirmed elevation in cancer tissue. The breast cancer mouse model, MMTV-PyMT (PyMT), developed breast tumors with lung metastasis; however, mice deleted in p38δ (PyMT/p38δ -/- ) exhibited delayed primary tumor formation and highly reduced lung metastatic burden. At the cellular level, we demonstrate that targeting of p38δ in breast cancer cells, MCF-7 and MDA-MB-231 resulted in a reduced rate of cell proliferation. In addition, cells lacking p38δ also displayed an increased cell-matrix adhesion and reduced cell detachment. This effect on cell adhesion was molecularly supported by the regulation of the focal adhesion kinase by p38δ in the human breast cell lines. These studies define a previously unappreciated role for p38δ in breast cancer development and evolution by regulating tumor growth and altering metastatic properties. This study proposes MAPK p38δ protein as a key factor in breast cancer. Lack of p38δ resulted in reduced primary tumor size and blocked the metastatic potential to the lungs.

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.

Science.gov (United States)

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

Casein Glycomacropeptide Hydrolysates Exert Cytoprotective Effect against Cellular Oxidative Stress by Up-Regulating HO-1 Expression in HepG2 Cells

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Tiange Li

2017-01-01

Full Text Available Oxidative stress is considered as an important mediator in the progression of metabolic disorders. The objective of this study was to investigate the potential hepatoprotective effects and mechanisms of bovine casein glycomacropeptide hydrolysates (GHP on hydrogen peroxide (H2O2-induced oxidative damage in HepG2 cells. Results showed that GHP significantly blocked H2O2-induced intracellular reactive oxygen species (ROS generation and cell viability reduction in a dose-dependent manner. Further, GHP concentration-dependently induced heme oxygenase-1 (HO-1 expression and increased nuclear factor-erythroid 2-related factor 2 (Nrf2 nuclear translocation. Moreover, pretreatment of GHP increased the activation of p38 mitogen-activated protein kinase (p38 MAPK and extracellular signal-regulated protein kinase 1/2 (ERK1/2, which were shown to contribute to Nrf2-mediated HO-1 expression. Taken together, GHP protected HepG2 cells from oxidative stress by activation of Nrf2 and HO-1 via p38 MAPK and ERK1/2 signaling pathways. Our findings indicate that bovine casein glycomacropeptide hydrolysates might be a potential ingredient in the treatment of oxidative stress-related disorders and further studies are needed to investigate the protective effects in vivo.

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.

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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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modulation of inflammation and pathology during dengue virus infection by p38 MAPK inhibitor SB203580.

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Fu, Yilong; Yip, Andy; Seah, Peck Gee; Blasco, Francesca; Shi, Pei-Yong; Hervé, Maxime

2014-10-01

Dengue virus (DENV) infection could lead to dengue fever (DF), dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The disease outcome is controlled by both viral and host factors. Inflammation mediators from DENV-infected cells could contribute to increased vascular permeability, leading to severe DHF/DSS. Therefore, suppression of inflammation could be a potential therapeutic approach for treatment of dengue patients. In this context, p38 MAPK (mitogen-activated protein kinase) is a key enzyme that modulates the initiation of stress and inflammatory responses. Here we show that SB203580, a p38 MAPK inhibitor, suppressed the over production of DENV-induced pro-inflammatory mediators such as TNF-α, IL-8, and RANTES from human PBMCs, monocytic THP-1, and granulocyte KU812 cell lines. Oral administration of SB203580 in DENV-infected AG129 mice prevented hematocrit rise and lymphopenia, limited the development of inflammation and pathology (including intestine leakage), and significantly improved survival. These results, for the first time, have provided experimental evidence to imply that a short term inhibition of p38 MAPK may be beneficial to reduce disease symptoms in dengue patients. Copyright © 2014 Elsevier B.V. All rights reserved.

Arctigenin induces apoptosis in colon cancer cells through ROS/p38MAPK pathway.

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Li, Qing-chun; Liang, Yun; Tian, Yuan; Hu, Guang-rui

2016-01-01

In the current study the antiproliferative effect of arctigenin, plant lignin, was evaluated on human colon cancer cell line HT-29. Furthermore, attempts were made to explore the signaling mechanism which may be responsible for its effect. Cell growth inhibition was assessed by MTT and LDH assays. Flow cytometric analysis was performed to determine cell arrest in the cell cycle phase and apoptosis. Furthermore, to confirm the apoptotic activity of arctigenin, caspase-9 and -3 activities analysis was performed. The levels of reactive oxygen species (ROS) and p38 mitogen activated protein kinase (MAPK) were investigated to determine their role in inducing apoptosis in arctigenin-treated HT-29 colon cancer cell line. MTT and LDH results demonstrated significant cell growth inhibitory effect of arctigenin on HT-29 cells in a dose-dependent manner. Furthermore, increase in cell number arrested at G2/M phase was observed in flow cytometric analysis upon arctigenin treatment. In addition, arctigenin increased the apoptotic ratio in a dose-dependent manner. The involvement of intrinsic apoptotic pathway was indicated by the activation of caspase-9 and -3. Moreover, increased ROS production, activation of p38 MAPK and changes in mitochondrial membrane potential (ΔΨm) also revealed the role of intrinsic apoptotic signaling pathway in cell growth inhibition after arctigenin exposure. Arctigenin induces apoptosis in HT-29 colon cancer cells by regulating ROS and p38 MAPK pathways.

Comparative Amino Acid Decomposition Analysis of Potent Type I P38α Inhibitors

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Ahmad Ebadi

2013-05-01

Full Text Available Background and purpose of the study:p38α is a member of mitogen-activated protein kinases (MAPK considered as a prominent target in development of anti-inflammatory agents. Any abnormality in the phosphorylation process leads to the different human diseases such as cancer, diabetes and inflammatory diseases. Several small molecule p38α inhibitors have been developed up to now. In this regard, structural elucidation of p38 inhibitors needs to be done enabling us in rational lead development strategies.Methods:Various interactions of three potent inhibitors with p38α active site have been evaluated in terms of binding energies and bond lengths via density function theory and MD simulations.Results:Our comparative study showed that both ab initio and MD simulation led to the relatively similar results in pharmacophore discrimination of p38α inhibitors.Conclusion:The results of the present study may find their usefulness in pharmacophore based modification of p38α inhibitors.

The Effects of Xiangqing Anodyne Spray on Treating Acute Soft-Tissue Injury Mainly Depend on Suppressing Activations of AKT and p38 Pathways

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Shudong Wang

2016-01-01

Full Text Available Objectives. In the present study we try to elucidate the mechanism of Xiangqing anodyne spray (XQAS effects on acute soft-tissue injury (STI. Methods. Acute STI model was established by hammer blow in the rat hind leg muscle. Within 8 hours, instantly after modeling and per 2-hour interval repeated topical applications with or without XQAS, CP or IH ethanol extracts spray (CPS and IHS were performed, respectively; muscle swelling rate and inflammation-related biochemical parameters, muscle histological observation, and mRNA and protein expression were then examined. Results. XQAS dose-dependently suppressed STI-caused muscle swelling, proinflammatory mediator productions, and oxidative stress as well as severe pathological changes in the injured muscle tissue. Moreover, CPS mainly by blocking p38 activation while IHS majorly by blocking AKT activation led to cytoplastic IκBα degradation with NF-κB p65 translocated into the nucleus. There are synergistic effects between CP and IH components in the XQAS on preventing from acute STI with suppressing IκBα degradation, NF-κB p65 translocation, and subsequent inflammation and oxidative stress-related abnormality. Conclusion. Marked effects of XQAS on treating acute STI are ascribed to strong anti-inflammatory and antioxidative actions with a reasonable combination of CP active components, blocking p38-NF-κB pathway activated, and IH active components, blocking AKT-NF-κB pathway activated.

Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage: Role of NF-κB, p38 and JNK MAPK pathway

International Nuclear Information System (INIS)

Ghosh, Jyotirmoy; Das, Joydeep; Manna, Prasenjit; Sil, Parames C.

2009-01-01

Cardiac dysfunction is a major cause of morbidity and mortality worldwide due to its complex pathogenesis. However, little is known about the mechanism of arsenic-induced cardiac abnormalities and the use of antioxidants as the possible protective agents in this pathophysiology. Conditionally essential amino acid, taurine, accounts for 25% to 50% of the amino acid pool in myocardium and possesses antioxidant properties. The present study has, therefore, been carried out to investigate the underlying mechanism of the beneficial role of taurine in arsenic-induced cardiac oxidative damage and cell death. Arsenic reduced cardiomyocyte viability, increased reactive oxygen species (ROS) production and intracellular calcium overload, and induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-κB (p65) phosphorylation. Pre-exposure of myocytes to an IKK inhibitor (PS-1145) prevented As-induced caspase-3 and PARP cleavage. Arsenic also markedly increased the activity of p38 and JNK MAPKs, but not ERK to that extent. Pre-treatment with SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated NF-κB and IKK phosphorylation indicating that p38 and JNK MAPKs are mainly involved in arsenic-induced NF-κB activation. Taurine treatment suppressed these apoptotic actions, suggesting that its protective role in arsenic-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways. Similarly, arsenic intoxication altered a number of biomarkers related to cardiac oxidative stress and other apoptotic indices in vivo and taurine supplementation could reduce it. Results suggest that taurine prevented arsenic-induced myocardial pathophysiology, attenuated NF-κB activation via IKK, p38 and JNK MAPK signaling pathways and could possibly provide a protection against As

Crosstalk between phospholipase D and sphingosine kinase in plant stress signaling

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Xuemin eWang

2012-03-01

Full Text Available The activation of phospholipase D (PLD produces phosphatidic acid (PA, whereas sphingosine kinase (SPHK phosphorylates long-chain bases (LCBs to generate LCB-1-phosphates (LCBPs such as phytosphingosine-1-phosphate (phyto-S1P. PA and phyto-S1P have been identified as lipid messengers. Recent studies have shown that PA interacts directly with SPHKs in Arabidopsis, and that the interaction promotes SPHK activity. However, SPHK and phyto-S1P act upstream of PLDα1 and PA in the stomatal response to abscisic acid (ABA. These findings indicate that SPHK/phyto-S1P and PLD/PA are co-dependent in the amplification of lipid messengers, and that crosstalk between the sphingolipid- and phospholipid-mediated signaling pathways may play important roles in plant stress signaling.

Inhibition of p38 MAPK enhances ABT-737-induced cell death in melanoma cell lines: novel regulation of PUMA.

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Keuling, Angela M; Andrew, Susan E; Tron, Victor A

2010-06-01

The mitogen-activated protein kinase (MAPK) pathway is constitutively activated in the majority of melanomas, promoting cell survival, proliferation and migration. In addition, anti-apoptotic Bcl-2 family proteins Mcl-1, Bcl-xL and Bcl-2 are frequently overexpressed, contributing to melanoma's well-documented chemoresistance. Recently, it was reported that the combination of MAPK pathway inhibition by specific MEK inhibitors and Bcl-2 family inhibition by BH3-mimetic ABT-737 synergistically induces apoptotic cell death in melanoma cell lines. Here we provide the first evidence that inhibition of another key MAPK, p38, synergistically induces apoptosis in melanoma cells in combination with ABT-737. We also provide novel mechanistic data demonstrating that inhibition of p38 increases expression of pro-apoptotic Bcl-2 protein PUMA. Furthermore, we demonstrate that PUMA can be cleaved by a caspase-dependent mechanism during apoptosis and identify what appears to be the PUMA cleavage product. Thus, our findings suggest that the combination of ABT-737 and inhibition of p38 is a promising, new treatment strategy that acts through a novel PUMA-dependent mechanism.

A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis

DEFF Research Database (Denmark)

Villumsen, Bine H; Danielsen, Jannie R; Povlsen, Lou

2013-01-01

uncover a new two-pronged signalling response, which by coupling p38-dependent phosphorylation with MIB1-catalysed ubiquitylation of ciliogenesis-promoting factors plays an important role in controlling centriolar satellite status and key centrosomal functions in a cell stress-regulated manner.......Centriolar satellites are small, granular structures that cluster around centrosomes, but whose biological function and regulation are poorly understood. We show that centriolar satellites undergo striking reorganization in response to cellular stresses such as UV radiation, heat shock......, and transcription blocks, invoking acute and selective displacement of the factors AZI1/CEP131, PCM1, and CEP290 from this compartment triggered by activation of the stress-responsive kinase p38/MAPK14. We demonstrate that the E3 ubiquitin ligase MIB1 is a new component of centriolar satellites, which interacts...

The Prognostic Role of NEDD9 and P38 Protein Expression Levels in Urinary Bladder Transitional Cell Carcinoma

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Ola A. Harb

2017-01-01

Full Text Available Background. The most common malignant tumor of the urinary bladder is transitional cell carcinoma (TCC. Neural precursor cell-expressed developmentally downregulated protein 9 (NEDD9 is found to be a cell adhesion mediator. P38 Mitogen-Activated Protein Kinase is a serine/threonine kinases member which can mediate carcinogenesis through intracellular signaling. Methods. To assess their prognostic role; NEDD9 and p38 protein were evaluated in sections from 50 paraffin blocks of TCC. Results. The high expressions of NEDD9 and p38 protein were significantly associated with grade, stage, distant metastasis (p<0.001, number of tumors, lymph node metastasis, and tumor size (p<0.001, 0.002; 0.018, <0.001; and 0.004, 0.007, respectively. High NEDD9 and p38 detection had a worse 3-year OS (p=0.041 and <0.001, respectively. By multivariate analysis the NEDD9 and p38 protein expression levels and various clinicopathological criteria including gender, grade, stage of the tumor, and regional lymph node involvement were independent prognostic parameters of TCC of the urinary bladder patients’ outcome. Conclusion. NEDD9 and p38 protein expressions were poor prognostic markers of TCC.

Adiponectin promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-27b through a CaMKII/AMPK/p38 signaling pathway in human chondrosarcoma cells.

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Huang, Chun-Yin; Chang, An-Chen; Chen, Hsien-Te; Wang, Shih-Wei; Lo, Yuan-Shun; Tang, Chih-Hsin

2016-09-01

Chondrosarcoma is the second most frequently occurring type of bone malignancy characterized by distant metastatic propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumour lymphangiogenesis and lymphatic metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. In recent years, adiponectin has also been indicated as facilitating tumorigenesis, angiogenesis and metastasis. However, the effect of adiponectin on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has remained largely a mystery. In the present study, we have shown a clinical correlation between adiponectin and VEGF-C, as well as tumour stage, in human chondrosarcoma tissues. We further demonstrated that adiponectin promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium from adiponectin-treated cells significantly induced tube formation and migration of human lymphatic endothelial cells. In addition, adiponectin knock down inhibited lymphangiogenesis in vitro and in vivo We also found that adiponectin-induced VEGF-C is mediated by the calmodulin-dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK) and p38 signaling pathway. Furthermore, the expression of miR-27b was negatively regulated by adiponectin via the CaMKII, AMPK and p38 cascade. The present study is the first to describe the mechanism of adiponectin-promoted lymphangiogenesis by up-regulating VEGF-C expression in chondrosarcomas. Thus, adiponectin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

EGCG-targeted p57/KIP2 reduces tumorigenicity of oral carcinoma cells: Role of c-Jun N-terminal kinase

International Nuclear Information System (INIS)

Yamamoto, Tetsuya; Digumarthi, Hari; Aranbayeva, Zina; Wataha, John; Lewis, Jill; Messer, Regina; Qin, Haiyan; Dickinson, Douglas; Osaki, Tokio; Schuster, George S.; Hsu, Stephen

2007-01-01

The green tea polyphenol epigallocatechin-3-gallate (EGCG) regulates gene expression differentially in tumor and normal cells. In normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. In humans, reduced expression of p57 often is associated with advanced tumors, and tumor cells with inactivated p57 undergo apoptosis when exposed to EGCG. The mechanism of p57 induction by EGCG is not well understood. Here, we show that in NHEK, EGCG-induces p57 via the p38 mitogen-activated protein kinase (MAPK) signaling pathway. In p57-negative tumor cells, JNK signaling mediates EGCG-induced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis via inhibition of c-Jun N-terminal kinase (JNK). We also found that restoration of p57 expression in tumor cells significantly reduced tumorigenicity in athymic mice. These results suggest that p57 expression may be an useful indicator for the clinical course of cancers, and could be potentially useful as a target for cancer therapies

Fluoxetine protects against methamphetamine‑induced lung inflammation by suppressing oxidative stress through the SERT/p38 MAPK/Nrf2 pathway in rats.

Science.gov (United States)

Wang, Yun; Gu, Yu-Han; Liu, Ming; Bai, Yang; Wang, Huai-Liang

2017-02-01

Methamphetamine (MA) abuse is a major public health and safety concern throughout the world and a growing burden on healthcare costs. The purpose of the present study was to investigate the protective effect of fluoxetine against MA‑induced chronic pulmonary inflammation and to evaluate the potential role of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidative stress. Wistar rats were divided into control, MA and two fluoxetine‑treated groups. Rats in the MA and the two fluoxetine‑treated groups were treated daily with intraperitoneal injection of 10 mg/kg MA twice daily. Rats in the two fluoxetine‑treated groups were injected intragastrically with fluoxetine (2 and 10 mg/kg) once daily, respectively. After 5 weeks, the rats were euthanized and hematoxylin and eosin staining, immunohistochemistry, western blot analysis and redox assay were performed. It was demonstrated that chronic exposure to MA can induce pulmonary inflammation in rats, with the symptoms of inflammatory cell infiltration, crowded lung parenchyma, thickened septum and a reduced number of alveolar sacs. Fluoxetine attenuated pulmonary inflammation and the expression of interleukin‑6 and tumor necrosis factor‑α in rat lungs. Fluoxetine inhibited MA‑induced increases in the expression levels of serotonin transporter (SERT) and p‑p38 mitogen‑activated protein kinase (MAPK), and reversed the MA‑induced decrease in nuclear Nrf2 and human heme oxygenase‑1 in lungs. Fluoxetine at 10 mg/kg significantly reversed the reduced glutathione (GSH) level, the ratio of GSH/oxidized glutathione, and the reactive oxygen species level in rat lungs from the MA group. These findings suggested that fluoxetine, a SERT inhibitor, has a protective effect against MA‑induced lung inflammation by suppressing oxidative stress through the SERT/p38 MAPK/Nrf2 pathway in rats.

Dioscin alleviates BDL- and DMN-induced hepatic fibrosis via Sirt1/Nrf2-mediated inhibition of p38 MAPK pathway

Energy Technology Data Exchange (ETDEWEB)

Gu, Lina; Tao, Xufeng; Xu, Youwei; Han, Xu; Qi, Yan; Xu, Lina; Yin, Lianhong; Peng, Jinyong, E-mail: jinyongpeng2014@163.com

2016-02-01

Oxidative stress is involved in hepatic stellate cells (HSCs) activation and extracellular matrix overproduction. We previously reported the promising effects of dioscin against CCl{sub 4}-induced liver fibrosis, but its effects and mechanisms on BDL- and DMN-induced liver fibrosis remain unknown. The results in the present study indicated that dioscin significantly inhibited HSCs activation and attenuated hepatic fibrosis in rats. Furthermore, dioscin markedly up-regulated the levels of sirtuin 1 (Sirt1), HO-1, GST, GCLC and GCLM via increasing the nuclear translocation of nuclear erythroid factor 2-related factor 2 (Nrf2), which in turn inhibited mitogen-activated protein kinase 14 (p38 MAPK) phosphorylation and reduced the levels of COL1A1, COL3A1, α-SMA and fibronectin. These results were further validated by knockdown of Sirt1 and Nrf2 using siRNAs silencing, and abrogation of p38 MAPK using SB-203580 (a p38 MAPK inhibitor) in HSC-T6 and LX-2 cells. Collectively, our findings confirmed the potent effects of dioscin against liver fibrosis and also provided novel insights into the mechanisms of this compound as a candidate for the prevention of liver fibrosis in the future. - Highlights: • Dioscin showed potent effects against BDL- and DMN-induced liver fibrosis in rats. • Dioscin significantly suppressed oxidative stress. • Dioscin triggered Sirt1/Nrf2-mediated inhibition of p38 MAPK pathway. • Dioscin should be developed as a novel candidate to treat liver fibrosis.

Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response

KAUST Repository

Almadanim, M. Cecí lia; Alexandre, Bruno M.; Rosa, Margarida T.G.; Sapeta, Helena; Leitã o, Antó nio E.; Ramalho, José C.; Lam, TuKiet T.; Negrã o, Só nia; Abreu, Isabel A.; Oliveira, M. Margarida

2017-01-01

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here

Pharmacologic ATM but not ATR kinase inhibition abrogates p21-dependent G1 arrest and promotes gastrointestinal syndrome after total body irradiation.

Science.gov (United States)

Vendetti, Frank P; Leibowitz, Brian J; Barnes, Jennifer; Schamus, Sandy; Kiesel, Brian F; Abberbock, Shira; Conrads, Thomas; Clump, David Andy; Cadogan, Elaine; O'Connor, Mark J; Yu, Jian; Beumer, Jan H; Bakkenist, Christopher J

2017-02-01

We show that ATM kinase inhibition using AZ31 prior to 9 or 9.25 Gy total body irradiation (TBI) reduced median time to moribund in mice to 8 days. ATR kinase inhibition using AZD6738 prior to TBI did not reduce median time to moribund. The striking finding associated with ATM inhibition prior to TBI was increased crypt loss within the intestine epithelium. ATM inhibition reduced upregulation of p21, an inhibitor of cyclin-dependent kinases, and blocked G1 arrest after TBI thereby increasing the number of S phase cells in crypts in wild-type but not Cdkn1a(p21 CIP/WAF1 )-/- mice. In contrast, ATR inhibition increased upregulation of p21 after TBI. Thus, ATM activity is essential for p21-dependent arrest while ATR inhibition may potentiate arrest in crypt cells after TBI. Nevertheless, ATM inhibition reduced median time to moribund in Cdkn1a(p21 CIP/WAF1 )-/- mice after TBI. ATM inhibition also increased cell death in crypts at 4 h in Cdkn1a(p21 CIP/WAF1 )-/-, earlier than at 24 h in wild-type mice after TBI. In contrast, ATR inhibition decreased cell death in crypts in Cdkn1a(p21 CIP/WAF1 )-/- mice at 4 h after TBI. We conclude that ATM activity is essential for p21-dependent and p21-independent mechanisms that radioprotect intestinal crypts and that ATM inhibition promotes GI syndrome after TBI.

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

Science.gov (United States)

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

2017-01-01

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

Requirement for Pectin Methyl Esterase and Preference for Fragmented over Native Pectins for Wall-associated Kinase-activated, EDS1/PAD4-dependent Stress Response in Arabidopsis*

Science.gov (United States)

Kohorn, Bruce D.; Kohorn, Susan L.; Saba, Nicholas J.; Martinez, Victoriano Meco

2014-01-01

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. PMID:24855660

Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.

Science.gov (United States)

Kohorn, Bruce D; Kohorn, Susan L; Saba, Nicholas J; Martinez, Victoriano Meco

2014-07-04

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Witters, L.A.; Bacon, G.W.

1985-01-01

The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

Energy Technology Data Exchange (ETDEWEB)

Liu, Jian-Zhong; Duan, Jicheng; Ni, Min; Liu, Zhen; Qiu, Wen-Li; Whitham, Steven A.; Qian, Wei-Jun

2017-09-29

classes of proteins, both in plants and in mammals, have been identified as targets of S-nitrosylation (5-9). In plants, proteins with diverse functions are S-nitrosylated at specific Cys residue(s) and their functions are either inhibited or enhanced by this modification (10-25). 3-Phosphoinositide-dependent protein kinase-1 (PDK1) and its downstream target, protein kinase B (PKB; also known as Akt), are central regulators of mammalian apoptosis (26-28). PKB is a member of the AGC family of protein kinases, which is activated by second messengers such as phospholipids and Ca2+ through PDK1. Mammalian PDK1 phosphorylates PKB to promote its function in suppressing programmed cell death (PCD) (27-30). PKB negatively regulates apoptosis by phosphorylation and inactivation of pro-apoptotic factors such as BAD and activation of anti-apoptotic factors such as CREB and IKK (27-29; and 31). Deficiency of the PDK1 gene(s) in Drosophila (32), mice (33), yeast (34-35) and tomato (36), respectively, results in lethality or severe apoptosis. PKB knockout mice display spontaneous apoptosis in several different tissues (37). In tomato, the PKB/Akt homolog, Adi3 (AvrPto-dependent Pto-interacting protein 3), physically interacts with and is phosphorylated by SlPDK1 (36). Silencing both SlPDK1 and Adi3 or treatment with a PDK1 inhibitor results in MAPKKK -dependent cell death, indicating that Adi3 functions analogously to the mammalian PKB/Akt by negatively regulating cell death via PDK1 phosphorylation (36). Yasukawa et al (38) showed that NO donors induced S-nitrosylation and inactivation of Akt/PKB kinase activity in vitro and in vivo and the mutant Akt1/PKB (C224S) was resistant to S-nitrosylation by NO and its kinase inactivation (38). Although the NO and PDK1-PKB/Akt pathways are both key regulators of cell death, the link between these two pathways has not been firmly established in plants. Here we show that the kinase activity of tomato SlPDK1 was inhibited by GSNO in a conce

The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells.

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Hongo, Akane; Okumura, Naoki; Nakahara, Makiko; Kay, EunDuck P; Koizumi, Noriko

2017-07-01

We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs). HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-β-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA. Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-β-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580. Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.

Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

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Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; Plaza-Davila, María; Martinez-Ruiz, Antonio; Fernandez-Bermejo, Miguel; Mateos-Rodriguez, Jose M; Salido, Gines M; Gonzalez, Antonio

2018-01-01

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca 2+ concentration ([Ca 2+ ] c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca 2+ ] c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells. © 2017 Wiley Periodicals, Inc.

A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils.

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Avdi, Natalie J; Malcolm, Kenneth C; Nick, Jerry A; Worthen, G Scott

2002-10-25

Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.

Modulation of fatty acid synthase degradation by concerted action of p38 MAP kinase, E3 ligase COP1, and SH2-tyrosine phosphatase Shp2.

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Yu, Jianxiu; Deng, Rong; Zhu, Helen H; Zhang, Sharon S; Zhu, Changhong; Montminy, Marc; Davis, Roger; Feng, Gen-Sheng

2013-02-08

The Src-homology 2 (SH2) domain-containing tyrosine phosphatase Shp2 has been known to regulate various signaling pathways triggered by receptor and cytoplasmic tyrosine kinases. Here we describe a novel function of Shp2 in control of lipid metabolism by mediating degradation of fatty acid synthase (FASN). p38-phosphorylated COP1 accumulates in the cytoplasm and subsequently binds FASN through Shp2 here as an adapter, leading to FASN-Shp2-COP1 complex formation and FASN degradation mediated by ubiquitination pathway. By fasting p38 is activated and stimulates FASN protein degradation in mice. Consistently, the FASN protein levels are dramatically elevated in mouse liver and pancreas in which Shp2/Ptpn11 is selectively deleted. Thus, this study identifies a new activity for Shp2 in lipid metabolism.

Attenuation of p38α MAPK stress response signaling delays the in vivo aging of skeletal muscle myofibers and progenitor cells.

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Papaconstantinou, John; Wang, Chen Z; Zhang, Min; Yang, San; Deford, James; Bulavin, Dmitry V; Ansari, Naseem H

2015-09-01

Functional competence and self-renewal of mammalian skeletal muscle myofibers and progenitor cells declines with age. Progression of the muscle aging phenotype involves the decline of juvenile protective factorsi.e., proteins whose beneficial functions translate directly to the quality of life, and self-renewal of progenitor cells. These characteristics occur simultaneously with the age-associated increase of p38α stress response signaling. This suggests that the maintenance of low levels of p38α activity of juvenile tissues may delay or attenuate aging. We used the dominant negative haploinsufficient p38α mouse (DN-p38α(AF/+)) to demonstrate that in vivo attenuation of p38α activity in the gastrocnemius of the aged mutant delays age-associated processes that include: a) the decline of the juvenile protective factors, BubR1, aldehyde dehydrogenase 1A (ALDH1A1), and aldehyde dehydrogenase 2 (ALDH2); b) attenuated expression of p16(Ink4a) and p19(Arf) tumor suppressor genes of the Cdkn2a locus; c) decreased levels of hydroxynonenal protein adducts, expression of COX2 and iNOS; d) decline of the senescent progenitor cell pool level and d) the loss of gastrocnemius muscle mass. We propose that elevated P-p38α activity promotes skeletal muscle aging and that the homeostasis of p38α impacts the maintenance of a beneficial healthspan.

Calcium-dependent protein kinase 21 phosphorylates 14-3-3 proteins in response to ABA signaling and salt stress in rice.

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Chen, Yixing; Zhou, Xiaojin; Chang, Shu; Chu, Zhilin; Wang, Hanmeng; Han, Shengcheng; Wang, Yingdian

2017-12-02

The calcium-dependent protein kinases (CDPKs) are a class of plant-specific kinase that directly bind Ca 2+ and mediate the calcium-signaling pathways to play important physiological roles in growth and development. The rice genome contains 31 CDPK genes, one of which, OsCPK21, is known to modulate the abscisic acid (ABA) and salt stress responses in this crop; however, the molecular mechanisms underlying this regulation are largely unknown. In the present study, we performed yeast two-hybrid screening, glutathione S-transferase pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays to confirm the interaction between OsCPK21 and one of its putative targets, Os14-3-3 (OsGF14e). We used an in vitro kinase assay and site-directed mutagenesis to verify that OsCPK21 phosphorylates OsGF14e at Tyr-138. We used real-time PCR to reveal that several ABA and salt inducible genes were more highly expressed in the OsCPK21-OE and OsGF14e WT-OE plants than in the mutant OsGF14e Y138A-OE and wild-type plants. These results suggest that OsCPK21 phosphorylates OsGF14e to facilitate the response to ABA and salt stress. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response

KAUST Repository

Almadanim, M. Cecília

2017-01-19

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analyzing OsCPK17 knockout, silencing, and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose phosphate synthase OsSPS4, and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

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

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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.

The p38/CYLD Pathway is Involved in Necroptosis Induced by Oxygen-glucose Deprivation Combined with ZVAD in Primary Cortical Neurons.

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Feng, Tao; Chen, WeiWei; Zhang, CaiYi; Xiang, Jie; Ding, HongMei; Wu, LianLian; Geng, DeQin

2017-08-01

Recently, necroptosis, a form of programmed necrosis, has been widely studied. It has previously been shown that knockout of lysine 63 deubiquitinase CYLD significantly inhibits necroptosis in other cell lines, and serum response factor (SRF) could regulate CYLD gene expression through p38 mitogen-activated protein kinase (p38 MAPK). In the following study, we show oxygen-glucose deprivation (OGD) combined with a caspase inhibitor, ZVAD (OGD/ZVAD), induced CYLD protein expression in a time-dependent manner. Immunofluorescence studies showed that CYLD was localized strongly to the nucleus and weakly to the cytoplasm of neurons. The expression of CYLD in the cytoplasm, but not in the nucleus, was increased significantly upon OGD treatment. SB203580 (a p38 MAPK inhibitor) protected against neuronal injury induced by OGD/ZVAD treatment. More importantly, SB203580 decreased CYLD protein levels by inhibiting SRF phosphorylation and indirectly prevented SRF from binding to a CYLD promoter. We also found that cells with knockdown of SRF by short interfering RNA in a lentivirus vector tolerated OGD/ZVAD-induced necroptosis, when the expression of CYLD protein decreased. The results show that SB203580 prevented necroptosis induced by OGD/ZVAD injury by blocking a p38/CYLD dependent pathway.

Seasonal variations of cellular stress response of the gilthead sea bream (Sparus aurata).

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Feidantsis, Konstantinos; Antonopoulou, Efthimia; Lazou, Antigone; Pörtner, Hans O; Michaelidis, Basile

2013-07-01

The present study aimed to investigate the seasonal cellular stress response in vital organs, like the heart, the liver, the whole blood and the skeletal (red and white) muscles of the Mediterranean fish Sparus aurata during a 1-year acclimatization period in the field, in two examined depths (0-2 m and 10-12 m). Processes studied included heat shock protein expression and protein kinase activation. Molecular responses were addressed through the expression of Hsp70 and Hsp90, the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK), the extracellular signal-regulated kinases (ERK-1/2) and c-Jun N-terminal kinases (JNK1/2/3). The induction of Hsp70 and Hsp90 and the phosphorylation of p38 MAPK, JNKs and ERKs in the examined five tissues of the gilthead sea bream indicated a cellular stress response under the prism of a seasonal pattern which was characterized by distinct tissue specificity. Specifically, Hsp induction and MAPK activation occurred before peak summer water temperatures, with no further increases in their levels despite increases in water temperatures. Moreover, although water temperature did not vary significantly with depth of immersion, significant effects of depth on cellular stress response were observed, probably caused by different light regime. The expression and the activation of these certain proteins can be used as tools to define the extreme thermal limits of the gilthead sea bream.

Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways.

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Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

2008-07-01

Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate. 2008 Wiley-Liss, Inc.

Fucoidan/FGF-2 induces angiogenesis through JNK- and p38-mediated activation of AKT/MMP-2 signalling

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Kim, Beom Su [Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830 (Korea, Republic of); Park, Ji-Yun [Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830 (Korea, Republic of); Kang, Hyo-Jin [Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Hyung-Jin [Department of Microbiology, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lee, Jun, E-mail: omslee@wku.ac.kr [Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830 (Korea, Republic of)

2014-08-08

Graphical abstract: Schematic diagram of the angiogenic activity mechanism by FGF-2/fucoidan treatment in HUVECs. Fucoidan enhances the FGF-2-induced phosphorylation of p38, JNK, and ERK MAPKs. However, p38 and JNK were involved in AKT phosphorylation and MMP-2 activation and resulted in enhanced angiogenic activity, such as tube formation and migration, in HUVECs. - Highlights: • The angiogenic activity of fucoidan in HUVECs was explored. • Fucoidan enhanced HUVEC proliferation, migration, and tube formation. • Fucoidan enhanced angiogenesis through p38 and JNK but not ERK in HUVECs. • Fucoidan targeted angiogenesis-mediated AKT/MMP-2 signalling in HUVECs. - Abstract: Angiogenesis is an important biological process in tissue development and repair. Fucoidan has previously been shown to potentiate in vitro tube formation in the presence of basic fibroblast growth factor (FGF-2). However, the underlying molecular mechanism remains largely unknown. This study was designed to investigate the action of fucoidan in angiogenesis in human umbilical vein endothelial cells (HUVECs) and to explore fucoidan-signalling pathways. First, we evaluated the effect of fucoidan on cell proliferation. Matrigel-based tube formation and wound healing assays were performed to investigate angiogenesis. Matrix metalloproteinase-2 (MMP-2) mRNA expression and activity levels were analysed by reverse transcription polymerase chain reaction (RT-PCR) and zymography, respectively. Additionally, phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) was detected by Western blot. The results indicate that fucoidan treatment significantly increased cell proliferation in the presence of FGF-2. Moreover, compared to the effect of FGF-2 alone, fucoidan and FGF-2 had a greater effect on tube formation and cell migration, and this effect was found to be synergistic. Furthermore, fucoidan enhanced the phosphorylation of extracellular signal-regulated kinase (ERK

Fucoidan/FGF-2 induces angiogenesis through JNK- and p38-mediated activation of AKT/MMP-2 signalling

International Nuclear Information System (INIS)

Kim, Beom Su; Park, Ji-Yun; Kang, Hyo-Jin; Kim, Hyung-Jin; Lee, Jun

2014-01-01

Graphical abstract: Schematic diagram of the angiogenic activity mechanism by FGF-2/fucoidan treatment in HUVECs. Fucoidan enhances the FGF-2-induced phosphorylation of p38, JNK, and ERK MAPKs. However, p38 and JNK were involved in AKT phosphorylation and MMP-2 activation and resulted in enhanced angiogenic activity, such as tube formation and migration, in HUVECs. - Highlights: • The angiogenic activity of fucoidan in HUVECs was explored. • Fucoidan enhanced HUVEC proliferation, migration, and tube formation. • Fucoidan enhanced angiogenesis through p38 and JNK but not ERK in HUVECs. • Fucoidan targeted angiogenesis-mediated AKT/MMP-2 signalling in HUVECs. - Abstract: Angiogenesis is an important biological process in tissue development and repair. Fucoidan has previously been shown to potentiate in vitro tube formation in the presence of basic fibroblast growth factor (FGF-2). However, the underlying molecular mechanism remains largely unknown. This study was designed to investigate the action of fucoidan in angiogenesis in human umbilical vein endothelial cells (HUVECs) and to explore fucoidan-signalling pathways. First, we evaluated the effect of fucoidan on cell proliferation. Matrigel-based tube formation and wound healing assays were performed to investigate angiogenesis. Matrix metalloproteinase-2 (MMP-2) mRNA expression and activity levels were analysed by reverse transcription polymerase chain reaction (RT-PCR) and zymography, respectively. Additionally, phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) was detected by Western blot. The results indicate that fucoidan treatment significantly increased cell proliferation in the presence of FGF-2. Moreover, compared to the effect of FGF-2 alone, fucoidan and FGF-2 had a greater effect on tube formation and cell migration, and this effect was found to be synergistic. Furthermore, fucoidan enhanced the phosphorylation of extracellular signal-regulated kinase (ERK

Helicobacter pylori-elicited induction in gastric mucosal matrix metalloproteinase-9 (MMP-9) release involves ERK-dependent cPLA2 activation and its recruitment to the membrane-localized Rac1/p38 complex.

Science.gov (United States)

Slomiany, B L; Slomiany, A

2016-06-01

Matrix metalloproteinases (MMPs) are a family of endopeptidases implicated in a wide rage of degenerative and inflammatory diseases, including Helicobacter pylori-associated gastritis, and gastric and duodenal ulcer. As gastric mucosal inflammatory responses to H. pylori are characterized by the rise in MMP-9 production, as well as the induction in mitogen-activated protein kinase (MAPK) and Rac1 activation, we investigated the role of Rac1/MAPK in the processes associated with the release of MMP-9. We show that H. pylori LPS-elicited induction in gastric mucosal MMP-9 release is associated with MAPK, ERK and p38 activation, and occurs with the involvement of Rac1 and cytosolic phospholipase A2 (cPLA2). Further, we demonstrate that the LPS-induced MMP-9 release requires ERK-mediated phosphorylation of cPLA2 on Ser(505) that is essential for its membrane localization with Rac1, and that this process necessitates p38 participation. Moreover, we reveal that the activation and membrane translocation of p38 to the Rac1-GTP complex plays a pivotal role in cPLA2-dependent enhancement in MMP-9 release. Hence, our findings provide a strong evidence for the role of ERK/cPLA2 and Rac1/p38/cPLA2 cascade in H. pylori LPS-induced up-regulation in gastric mucosal MMP-9 release.

Duration of streptozotocin-induced diabetes differentially affects p38-mitogen-activated protein kinase (MAPK phosphorylation in renal and vascular dysfunction

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Gupta Akanksha

2005-03-01

Full Text Available Abstract Background In the present study we tested the hypothesis that progression of streptozotocin (STZ-induced diabetes (14-days to 28-days would produce renal and vascular dysfunction that correlate with altered p38- mitogen-activated protein kinase (p38-MAPK phosphorylation in kidneys and thoracic aorta. Methods Male Sprague Dawley rats (350–400 g were randomized into three groups: sham (N = 6, 14-days diabetic (N = 6 and 28-days diabetic rats (N = 6. Diabetes was induced using a single tail vein injection of STZ (60 mg/kg, I.V. on the first day. Rats were monitored for 28 days and food, water intake and plasma glucose levels were noted. At both 14-days and 28-days post diabetes blood samples were collected and kidney cortex, medulla and aorta were harvested from each rat. Results The diabetic rats lost body weight at both 14-days (-10% and 28-days (-13% more significantly as compared to sham (+10% group. Glucose levels were significantly elevated in the diabetic rats at both 14-days and 28-days post-STZ administration. Renal dysfunction as evidenced by renal hypertrophy, increased plasma creatinine concentration and reduced renal blood flow was observed in 14-days and 28-days diabetes. Vascular dysfunction as evidenced by decreased carotid blood flow was observed in 14-days and 28-days diabetes. We observed an up-regulation of inducible nitric oxide synthase (iNOS, prepro endothelin-1 (preproET-1 and phosphorylated p38-MAPK in thoracic aorta and kidney cortex but not in kidney medulla in 28-days diabetes group. Conclusion The study provides evidence that diabetes produces vascular and renal dysfunction with a profound effect on signaling mechanisms at later stage of diabetes.

Cyclin D1 represses p300 transactivation through a cyclin-dependent kinase-independent mechanism.

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Fu, Maofu; Wang, Chenguang; Rao, Mahadev; Wu, Xiaofang; Bouras, Toula; Zhang, Xueping; Li, Zhiping; Jiao, Xuanmao; Yang, Jianguo; Li, Anping; Perkins, Neil D; Thimmapaya, Bayar; Kung, Andrew L; Munoz, Alberto; Giordano, Antonio; Lisanti, Michael P; Pestell, Richard G

2005-08-19

Cyclin D1 encodes a regulatory subunit, which with its cyclin-dependent kinase (Cdk)-binding partner forms a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. In addition to its Cdk binding-dependent functions, cyclin D1 regulates cellular differentiation in part by modifying several transcription factors and nuclear receptors. The molecular mechanism through which cyclin D1 regulates the function of transcription factors involved in cellular differentiation remains to be clarified. The histone acetyltransferase protein p300 is a co-integrator required for regulation of multiple transcription factors. Here we show that cyclin D1 physically interacts with p300 and represses p300 transactivation. We demonstrated further that the interaction of the two proteins occurs at the peroxisome proliferator-activated receptor gamma-responsive element of the lipoprotein lipase promoter in the context of the local chromatin structure. We have mapped the domains in p300 and cyclin D1 involved in this interaction. The bromo domain and cysteine- and histidine-rich domains of p300 were required for repression by cyclin D1. Cyclin D1 repression of p300 was independent of the Cdk- and retinoblastoma protein-binding domains of cyclin D1. Cyclin D1 inhibits histone acetyltransferase activity of p300 in vitro. Microarray analysis identified a signature of genes repressed by cyclin D1 and induced by p300 that promotes cellular differentiation and induces cell cycle arrest. Together, our results suggest that cyclin D1 plays an important role in cellular proliferation and differentiation through regulation of p300.

EXPRESSION OF CALCIUM-DEPENDENT PROTEIN KINASE (CDPK GENES IN VITIS AMURENSIS UNDER ABIOTIC STRESS CONDITIONS

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Dubrovina A.S.

2012-08-01

Full Text Available Abiotic stresses, such as extreme temperatures, soil salinity, or water deficit, are one of the major limiting factors of crop productivity worldwide. Examination of molecular and genetic mechanisms of abiotic stress tolerance in plants is of great interest to plant biologists. Calcium-dependent protein kinases (CDPKs, which are the most important Ca2+ sensors in plants, are known to play one of the key roles in plant adaptation to abiotic stress. CDPK is a multigene family of enzymes. Analysis of CDPK gene expression under various abiotic stress conditions would help identify those CDPKs that might play important roles in plant adaptation to abiotic stress. We focused on studying CDPK gene expression under osmotic, water deficit, and temperature stress conditions in a wild-growing grapevine Vitis amurensis Rurp., which is native to the Russian Far East and is known to possess high adaptive potential and high level of resistance against adverse environmental conditions. Healthy V. amurensis cuttings (excised young stems with one healthy leaf were used for the treatments. For the non-stress treatment, we placed the cuttings in distilled water for 12 h at room temperature. For the water-deficit stress, detached cuttings were laid on a paper towel for 12 h at room temperature. For osmotic stress treatments, the cuttings were placed in 0.4 М NaCl and 0.4 М mannitol solutions for 12 h at room temperature. To examine temperature stress tolerance, the V. amurensis cuttings were placed in a growth chamber at +10oC and +37oC for 12 h. The total expression of VaCDPK genes was examined by semiquantitative RT-PCR with degenerate primers designed to the CDPK kinase domain. The total level of CDPK gene expression increased under salt and decreased under low temperature stress conditions. We sequenced 300 clones of the amplified part of different CDPK transcripts obtained from the analyzed cDNA probes. Analysis of the cDNA sequences identified 8 different

Concurrent suppression of NF-κB, p38 MAPK and reactive oxygen species formation underlies the effect of a novel compound isolated from Curcuma comosa Roxb. in LPS-activated microglia.

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Jiamvoraphong, Nittaya; Jantaratnotai, Nattinee; Sanvarinda, Pantip; Tuchinda, Patoomratana; Piyachaturawat, Pawinee; Thampithak, Anusorn; Sanvarinda, Pimtip

2017-07-01

We investigated the molecular mechanisms underlying the effect of (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol, also known as compound 092, isolated from Curcuma comosa Roxb on the production of pro-inflammatory mediators and oxidative stress in lipopolysaccharide (LPS)-activated highly aggressive proliferating immortalized (HAPI) microglial cell lines. Nitric oxide (NO) production was determined using the Griess reaction, and reverse transcription polymerase chain reaction was used to measure the expression of inducible nitric oxide synthase (iNOS) mRNA. Western blotting was used to determine the levels of pro-inflammatory mediators and their related upstream proteins. Compound 092 suppressed NO production and iNOS expression in LPS-stimulated HAPI cells. These effects originated from the ability of compound 092 to attenuate the activation of nuclear factor (NF)-κB as determined by the reduction in p-NF-κB and p-IκB kinase (IKK) protein levels. Compound 092 also significantly lowered LPS-activated intracellular reactive oxygen species production and p38 mitogen-activated protein kinase (MAPK) activation. Compound 092 suppresses microglial activation through attenuation of p38 MAPK and NF-κB activation. Compound 092 thus holds the potential to treat neurodegenerative disorders associated with neuroinflammation and oxidative stress. © 2017 Royal Pharmaceutical Society.

Inhibition of cyclophilin A suppresses H2O2-enhanced replication of HCMV through the p38 MAPK signaling pathway.

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Xiao, Jun; Song, Xin; Deng, Jiang; Lv, Liping; Ma, Ping; Gao, Bo; Zhou, Xipeng; Zhang, Yanyu; Xu, Jinbo

2016-09-01

Human cytomegalovirus (HCMV) infection can be accelerated by intracellular and extracellular hydrogen peroxide (H2O2) stimulation, mediated by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. However, it remains unknown whether host gene expression is involved in H2O2-upregulated HCMV replication. Here, we show that the expression of the host gene, cyclophilin A (CyPA), could be facilitated by treatment with H2O2 in a dose-dependent manner. Experiments with CyPA-specific siRNA, or with cyclosporine A, an inhibitor of CyPA, confirmed that H2O2-mediated upregulation of HCMV replication is specifically mediated by upregulation of CyPA expression. Furthermore, depletion or inhibition of CyPA reduced H2O2-induced p38 activation, consistent with that of H2O2-upregulated HCMV lytic replication. These results show that H2O2 is capable of activating ROS-CyPA-p38 MAPK interactions to enhance HCMV replication.

dependent/calmodulin- stimulated protein kinase from moss

Indian Academy of Sciences (India)

Unknown

stimulated protein kinase; CDPK, calmodulin domain-like protein kinase; KM14, 14 amino acid synthetic peptide; .... used were obtained from Sigma Chemical Company, USA, ..... Plant chimeric Ca2+/Calmodulin-dependent protein kinase.

Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

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Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

2009-06-01

Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM. (c) 2009 Wiley-Liss, Inc.

Osmostress induces autophosphorylation of Hog1 via a C-terminal regulatory region that is conserved in p38α.

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Inbal Maayan

Full Text Available Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks. Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it's removal from p38α abolishes p38α's autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.

Osmostress induces autophosphorylation of Hog1 via a C-terminal regulatory region that is conserved in p38α.

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Maayan, Inbal; Beenstock, Jonah; Marbach, Irit; Tabachnick, Shira; Livnah, Oded; Engelberg, David

2012-01-01

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it's removal from p38α abolishes p38α's autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.

TGF-beta1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k.

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Lechuga, Carmen G; Hernández-Nazara, Zamira H; Domínguez Rosales, José-Alfredo; Morris, Elena R; Rincón, Ana Rosa; Rivas-Estilla, Ana María; Esteban-Gamboa, Andrés; Rojkind, Marcos

2004-11-01

Transforming growth factor-beta1 (TGF-beta1), the main cytokine involved in liver fibrogenesis, induces expression of the type I collagen genes in hepatic stellate cells by a transcriptional mechanism, which is hydrogen peroxide and de novo protein synthesis dependent. Our recent studies have revealed that expression of type I collagen and matrix metalloproteinase-13 (MMP-13) mRNAs in hepatic stellate cells is reciprocally modulated. Because TGF-beta1 induces a transient elevation of alpha1(I) collagen mRNA, we investigated whether this cytokine was able to induce the expression of MMP-13 mRNA during the downfall of the alpha1(I) collagen mRNA. In the present study, we report that TGF-beta1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of alpha1(I) collagen mRNA. This change in MMP-13 mRNA expression occurs within the first 6 h postcytokine administration and is accompanied by a twofold increase in gene transcription and a fivefold decrease in mRNA half-life. This is followed by increased expression of MMP-13 mRNA, which reaches maximal values by 48 h. Our results also show that this TGF-beta1-mediated effect is de novo protein synthesis-dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70(S6k). Altogether, our data suggest that regulation of MMP-13 by TGF-beta1 is a complex process involving transcriptional and posttranscriptional mechanisms.

Comparative analysis of Homo sapiens and Mus musculus cyclin-dependent kinase (CDK) inhibitor genes p16 (MTS1) and p15 (MTS2).

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Jiang, P; Stone, S; Wagner, R; Wang, S; Dayananth, P; Kozak, C A; Wold, B; Kamb, A

1995-12-01

Cyclin-dependent kinase inhibitors are a growing family of molecules that regulate important transitions in the cell cycle. At least one of these molecules, p16, has been implicated in human tumorigenesis while its close homolog, p15, is induced by cell contact and transforming growth factor-beta (TGF-beta). To investigate the evolutionary and functional features of p15 and p16, we have isolated mouse (Mus musculus) homologs of each gene. Comparative analysis of these sequences provides evidence that the genes have similar functions in mouse and human. In addition, the comparison suggests that a gene conversion event is part of the evolution of the human p15 and p16 genes.

The yeast Sks1p kinase signaling network regulates pseudohyphal growth and glucose response.

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Cole Johnson

2014-03-01

Full Text Available The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans.

A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

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Anna H. Y. Law

2013-04-01

Full Text Available Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF-alpha through p38 mitogen activated protein kinase (MAPK. However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1 and protein phosphatase type 2A (PP2A in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

Kinase inhibitors: a new class of antirheumatic drugs

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Kyttaris VC

2012-09-01

Full Text Available Vasileios C KyttarisDivision of Rheumatology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USAAbstract: The outlook for patients with rheumatoid arthritis has improved significantly over the last three decades with the use of disease-modifying antirheumatic drugs. However, despite the use of methotrexate, cytokine inhibitors, and molecules targeting T and B cells, a percentage of patients do not respond or lose their response over time. The autoimmune process in rheumatoid arthritis depends on activation of immune cells, which utilize intracellular kinases to respond to external stimuli such as cytokines, immune complexes, and antigens. In the past decade, small molecules targeting several kinases, such as p38 MAPK, Syk, and JAK have been developed. Several p38 MAPK inhibitors proved ineffective in treating rheumatoid arthritis. The Syk inhibitor, fostamatinib, proved superior to placebo in Phase II trials and is currently under Phase III investigation. Tofacitinib, a JAK1/3 inhibitor, was shown to be efficacious in two Phase III trials, while VX-509, a JAK3 inhibitor, showed promising results in a Phase II trial. Fostamatinib and tofacitinib were associated with increased rates of infection, elevation of liver enzymes, and neutropenia. Moreover, fostamatinib caused elevations of blood pressure and diarrhea, while tofacitinib was associated with an increase in creatinine and elevation of lipid levels.Keywords: rheumatoid arthritis, kinase inhibitors, mitogen-activated phosphokinase p38, spleen tyrosine kinase, Janus kinases

Novel adenosine 3',5'-cyclic monophosphate dependent protein kinases in a marine diatom

International Nuclear Information System (INIS)

Lin, P.P.C.; Volcani, B.E.

1989-01-01

Two novel adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg 2+ and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser( 32 P)-Ser-Asn-Ala-Arg and have an apparent M r of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M r of about 78,000 is photolabeled with 8-azido[ 32 P]cAMP and is also phosphorylated with [γ- 32 P]ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids

A Novel Hydroxamate-Based Compound WMJ-J-09 Causes Head and Neck Squamous Cell Carcinoma Cell Death via LKB1-AMPK-p38MAPK-p63-Survivin Cascade.

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Yen, Chia-Sheng; Choy, Cheuk-Sing; Huang, Wei-Jan; Huang, Shiu-Wen; Lai, Pin-Ye; Yu, Meng-Chieh; Shiue, Ching; Hsu, Ya-Fen; Hsu, Ming-Jen

2018-01-01

Growing evidence shows that hydroxamate-based compounds exhibit broad-spectrum pharmacological properties including anti-tumor activity. However, the precise mechanisms underlying hydroxamate derivative-induced cancer cell death remain incomplete understood. In this study, we explored the anti-tumor mechanisms of a novel aliphatic hydroxamate-based compound, WMJ-J-09, in FaDu head and neck squamous cell carcinoma (HNSCC) cells. WMJ-J-09 induced G2/M cell cycle arrest and apoptosis in FaDu cells. These actions were associated with liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38MAPK) activation, transcription factor p63 phosphorylation, as well as modulation of p21 and survivin. LKB1-AMPK-p38MAPK signaling blockade reduced WMJ-J-09's enhancing effects in p63 phosphorylation, p21 elevation and survivin reduction. Moreover, WMJ-J-09 caused an increase in α-tubulin acetylation and interfered with microtubule assembly. Furthermore, WMJ-J-09 suppressed the growth of subcutaneous FaDu xenografts in vivo . Taken together, WMJ-J-09-induced FaDu cell death may involve LKB1-AMPK-p38MAPK-p63-survivin signaling cascade. HDACs inhibition and disruption of microtubule assembly may also contribute to WMJ-J-09's actions in FaDu cells. This study suggests that WMJ-J-09 may be a potential lead compound and warrant the clinical development in the treatment of HNSCC.

Calcium-dependent but calmodulin-independent protein kinase from soybean

International Nuclear Information System (INIS)

Harmon, A.C.; Putnam-Evans, C.; Cormier, M.J.

1987-01-01

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≥ 2 micromolar). The protein kinase activity was stimulated 100-fold by ≥ 10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤ 2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound 45 Ca 2+ in the presence of KCl and MgCl 2 , which indicated that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity

Pratol, an O-Methylated Flavone, Induces Melanogenesis in B16F10 Melanoma Cells via p-p38 and p-JNK Upregulation

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You Chul Chung

2017-10-01

Full Text Available Tyrosinase is the rate-limiting enzyme critical for melanin synthesis. It controls pigmentation in the skin. Activation of tyrosinase is currently the most common approach in the development of tanning and haircare products. Pratol is a 7-hydroxy-4-methoxyflavone found in Trifolium pratense. In this study, we investigated the effects of pratol on melanogenesis. We also studied the mechanism of action of pratol in B16F10 mouse melanoma cells. The cells were treated with various concentrations (6.25, 12.5, 25, and 50 μM of pratol to observe its effects. The results showed that pratol significantly increased melanin content and tyrosinase activity in the cells without being cytotoxic. In addition, pratol strongly increased the expression of tyrosinase and tyrosinase-related protein-1 and 2 by enhancing the expression of microphthalmia-associated transcription factor. Furthermore, pratol stimulated melanogenesis via the phosphorylation of p38, c-Jun N-terminal kinases (JNK, and extracellular signal–regulated kinase (ERK. The findings from an assay searching for the inhibitor revealed that SB203580 (a specific p38 inhibitor or SP600125 (a p-JNK inhibitor attenuated pratol-induced cellular tyrosinase activity whereas PD98059 (an ERK inhibitor did not. Additionally, pratol interfered with the phosphorylation of p-AKT. We also found that pratol-induced melanogenesis was reversed by H89, which is a specific protein kinase A inhibitor. The results suggest that, owing to its multi-functional properties, pratol may be a potential tanning agent or a therapeutic agent for hair depigmentation in the cosmetic industry.

Enhanced expression of WD repeat-containing protein 35 (WDR35 stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation

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Tsunekawa Koji

2013-01-01

Full Text Available Abstract Background Domoic acid (DA is an excitatory amino acid analogue of kainic acid (KA that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo. Results Our results showed that intraperitoneally administered DA was present in the brain and induced neurodegenerative changes including apoptosis in the CA1 region of the hippocampus. DA also increased the expression of WDR35 mRNA and protein in a dose- and time-dependent manner in the hippocampus. In experiments using glutamate receptor antagonists, the AMPA/KA receptor antagonist NBQX significantly attenuated the DA-induced increase in WDR35 protein expression, but the NMDA receptor antagonist MK-801 did not. In addition, the radical scavenger edaravone significantly attenuated the DA-induced increase in WDR35 protein expression. Furthermore, NBQX and edaravone significantly attenuated the DA-induced increase in p38 MAPK phosphorylation. Conclusion In summary, our results indicated that DA activated AMPA/KA receptors and induced ROS production and p38 MAPK phosphorylation, resulting in an increase in the expression of WDR35 in vivo.

Disruption of Smad-dependent signaling for growth of GST-P-positive lesions from the early stage in a rat two-stage hepatocarcinogenesis model

International Nuclear Information System (INIS)

Ichimura, Ryohei; Mizukami, Sayaka; Takahashi, Miwa; Taniai, Eriko; Kemmochi, Sayaka; Mitsumori, Kunitoshi; Shibutani, Makoto

2010-01-01

To clarify the involvement of signaling of transforming growth factor (TGF)-β during the hepatocarcinogenesis, the immunohistochemical distribution of related molecules was analyzed in relation with liver cell lesions expressing glutathione S-transferase placental form (GST-P) during liver tumor promotion by fenbendazole, phenobarbital, piperonyl butoxide, or thioacetamide, using rats. Our study focused on early-stage promotion (6 weeks after starting promotion) and late-stage promotion (57 weeks after starting promotion). With regard to Smad-dependent signaling, cytoplasmic accumulation of phosphorylated Smad (phospho-Smad)-2/3 - identified as Smad3 by later immunoblot analysis - increased in the subpopulation of GST-P + foci, while Smad4, a nuclear transporter of Smad2/3, decreased during early-stage promotion. By late-stage promotion, GST-P + lesions lacking phospho-Smad2/3 had increased in accordance with lesion development from foci to carcinomas, while Smad4 largely disappeared in most proliferative lesions. With regard to Smad-independent mitogen-activated protein kinases, GST-P + foci that co-expressed phospho-p38 mitogen-activated protein kinase increased during early-stage promotion; however, p38-downstream phospho-activating transcriptional factor (ATF)-2, ATF3, and phospho-c-Myc, were inversely downregulated without relation to promotion. By late-stage promotion, proliferative lesions downregulated phospho-ATF2 and phospho-c-Myc along with lesion development, as with downregulation of phospho-p38 in all lesions. These results suggest that from the early stages, carcinogenic processes were facilitated by disruption of tumor suppressor functions of Smad-dependent signaling, while Smad-independent activation of p38 was an early-stage phenomenon. GST-P - foci induced by promotion with agonists of peroxisome proliferator-activated receptor-α did not change Smad expression, suggesting an aberration in the Smad-dependent signaling prerequisites for induction

Lipoxin A4 regulates expression of the estrogen receptor and inhibits 17β-estradiol induced p38 mitogen-activated protein kinase phosphorylation in human endometriotic stromal cells.

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Chen, Shuo; Wu, Rong-Feng; Su, Lin; Zhou, Wei-Dong; Zhu, Mao-Bi; Chen, Qiong-Hua

2014-07-01

To study the role of lipoxin A4 (LXA4) in endometriosis. Molecular analysis in human samples and primary human endometriotic stromal cells (ESCs). University hospital. Forty-nine premenopausal women (30 patients with endometriosis and 19 controls). Normal and ectopic endometrial biopsies obtained during surgery performed during the proliferative phase of the menstrual cycle; ESCs used for in vitro studies. Levels of LXA4 measured by enzyme-linked immunosorbent assay (ELISA); mRNA levels of the estrogen receptor (ER), progestogen receptor (PR), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR); and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation evaluated by Western blotting. The LXA4 expression level decreased in ectopic tissue as well as ERα and PR, although the expression of ERβ increased in ectopic endometrium compared with the controls. Investigations with correlation analysis revealed the expression of LXA4 was positively correlated with ERα and negatively correlated with ERβ in vivo. Moreover, administering LXA4 could augment ERβ expression in ESCs and inhibit the 17β-estradiol-induced phosphorylation of p38 MAPK very likely through ERβ. Our findings indicate that LXA4 regulates ERβ expression and inhibits 17β-estradiol-induced phosphorylation of p38 MAPK, very likely through ERβ in ESCs. Copyright © 2014. Published by Elsevier Inc.

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

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Daniel Thomas

Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

Ampelopsin-induced reactive oxygen species enhance the apoptosis of colon cancer cells by activating endoplasmic reticulum stress-mediated AMPK/MAPK/XAF1 signaling

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Park, Ga Bin; Jeong, Jee-Yeong; Kim, Daejin

2017-01-01

Ampelopsin (Amp) is bioactive natural product and exerts anti-cancer effects against several cancer types. The present study investigated the anti-colon cancer activity of Amp and explored its mechanism of action. The treatment of colon cancer cells with Amp resulted in the dose- and time-dependent induction of apoptosis via the activation of endoplasmic reticulum (ER) stress, 5′ adenosine monophosphate-activated protein kinase (AMPK), and c-Jun N-terminal protein kinase (JNK)/p38 mitogen-activated protein kinases (MAPKs). Salubrinal, an ER stress inhibitor, prevented the upregulation of ER stress-associated proteins, including phosphorylated protein kinase RNA-like ER kinase, phosphorylated eukaryotic translation initiation factor 2α, glucose-regulated protein 78, and CCAAT/enhancer-binding protein homologous protein, as well as suppressing AMPK activation and the MAPK signaling pathway. Knockdown of AMPK by RNA interference failed to block ER stress. Additionally, SP600125 (a JNK inhibitor) and SB203580 (a p38-MAPK inhibitor) effectively inhibited apoptosis and attenuated the expression of X-linked IAP-associated factor 1 (XAF1) and apoptotic Bcl-2 family proteins (BCL2 antagonist/killer 1 and BCL2-associated X protein) in Amp-treated colon cancer cells. Furthermore, reactive oxygen species (ROS)-mediated ER stress/AMPK apoptotic signaling pathway in Amp-treated colon cancer cells were markedly inhibited by treatment with N-acetyl-L-cysteine, a ROS scavenger. These results demonstrate that treatment with Amp induces the apoptotic death of colon cancer cells through ER stress-initiated AMPK/MAPK/XAF1 signaling. These results also provide experimental information for developing Amp as therapeutic drug against colon cancer. PMID:29250183

Cisplatin Induces Cytotoxicity through the Mitogen-Activated Protein Kinase Pathways ana Activating Transcription Factor 3

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Carly St. Germain

2010-07-01

Full Text Available The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3 as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogenactivated protein kinase (MAPK pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellularsignal-regulated kinase, and p38 resulted in decreasedATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-ylF2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/murine embryonic fibroblasts (MEFs were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects.

Inducible Knockout of the Cyclin-Dependent Kinase 5 Activator p35 Alters Hippocampal Spatial Coding and Neuronal Excitability

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Eriko Kamiki

2018-05-01

Full Text Available p35 is an activating co-factor of Cyclin-dependent kinase 5 (Cdk5, a protein whose dysfunction has been implicated in a wide-range of neurological disorders including cognitive impairment and disease. Inducible deletion of the p35 gene in adult mice results in profound deficits in hippocampal-dependent spatial learning and synaptic physiology, however the impact of the loss of p35 function on hippocampal in vivo physiology and spatial coding remains unknown. Here, we recorded CA1 pyramidal cell activity in freely behaving p35 cKO and control mice and found that place cells in the mutant mice have elevated firing rates and impaired spatial coding, accompanied by changes in the temporal organization of spiking both during exploration and rest. These data shed light on the role of p35 in maintaining cellular and network excitability and provide a physiological correlate of the spatial learning deficits in these mice.

Inducible Knockout of the Cyclin-Dependent Kinase 5 Activator p35 Alters Hippocampal Spatial Coding and Neuronal Excitability

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Kamiki, Eriko; Boehringer, Roman; Polygalov, Denis; Ohshima, Toshio; McHugh, Thomas J.

2018-01-01

p35 is an activating co-factor of Cyclin-dependent kinase 5 (Cdk5), a protein whose dysfunction has been implicated in a wide-range of neurological disorders including cognitive impairment and disease. Inducible deletion of the p35 gene in adult mice results in profound deficits in hippocampal-dependent spatial learning and synaptic physiology, however the impact of the loss of p35 function on hippocampal in vivo physiology and spatial coding remains unknown. Here, we recorded CA1 pyramidal cell activity in freely behaving p35 cKO and control mice and found that place cells in the mutant mice have elevated firing rates and impaired spatial coding, accompanied by changes in the temporal organization of spiking both during exploration and rest. These data shed light on the role of p35 in maintaining cellular and network excitability and provide a physiological correlate of the spatial learning deficits in these mice. PMID:29867369

The role of p38 in mitochondrial respiration in male and female mice.

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Ju, Xiaohua; Wen, Yi; Metzger, Daniel; Jung, Marianna

2013-06-07

p38 is a mitogen-activated protein kinase and mediates cell growth, cell differentiation, and synaptic plasticity. The aim of this study is to determine the extent to which p38 plays a role in maintaining mitochondrial respiration in male and female mice under a normal condition. To achieve this aim, we have generated transgenic mice that lack p38 in cerebellar Purkinje neurons by crossing Pcp2 (Purkinje cell protein 2)-Cre mice with p38(loxP/loxP) mice. Mitochondria from cerebellum were then isolated from the transgenic and wild-type mice to measure mitochondrial respiration using XF24 respirometer. The mRNA and protein expression of cytochrome c oxidase (COX) in cerebellum were also measured using RT-PCR and immunoblot methods. Separately, HT22 cells were used to determine the involvement of 17β-estradiol (E2) and COX in mitochondrial respiration. The genetic knockout of p38 in Purkinje neurons suppressed the mitochondrial respiration only in male mice and increased COX expression only in female mice. The inhibition of COX by sodium azide (SA) sharply suppressed mitochondrial respiration of HT22 cells in a manner that was protected by E2. These data suggest that p38 is required for the mitochondrial respiration of male mice. When p38 is below a normal level, females may maintain mitochondrial respiration through COX up-regulation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

p38α MAPK Is Required for Tooth Morphogenesis and Enamel Secretion*

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Greenblatt, Matthew B.; Kim, Jung-Min; Oh, Hwanhee; Park, Kwang Hwan; Choo, Min-Kyung; Sano, Yasuyo; Tye, Coralee E.; Skobe, Ziedonis; Davis, Roger J.; Park, Jin Mo; Bei, Marianna; Glimcher, Laurie H.; Shim, Jae-Hyuck

2015-01-01

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678–27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38αK14 mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and β4-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel. PMID:25406311

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

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Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Crystal structure of human cyclin-dependent kinase-2 complex with MK2 inhibitor TEI-I01800: insight into the selectivity

Energy Technology Data Exchange (ETDEWEB)

Fujino, Aiko; Fukushima, Kei; Kubota, Takaharu; Kosugi, Tomomi; Takimoto-Kamimura, Midori, E-mail: m.kamimura@teijin.co.jp [Teijin Pharma Limited, 4-3-2 Asahigaoka, Hino-shi, Tokyo 191-8512 (Japan)

2013-11-01

The Gly-rich loop of cyclin-dependent kinase 2 (CDK2) bound to TEI-I01800 as an MK2 specific inhibitor forms a β-sheet which is a common structure in CDK2–ligand complexes. Here, the reason why TEI-I01800 does not become a strong inhibitor against CDK2 based on the conformation of TEI-I01800 is presented. Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2) is a Ser/Thr kinase from the p38 mitogen-activated protein kinase signalling pathway and plays an important role in inflammatory diseases. The crystal structure of the MK2–TEI-I01800 complex has been reported; its Gly-rich loop was found to form an α-helix, not a β-sheet as has been observed for other Ser/Thr kinases. TEI-I01800 is 177-fold selective against MK2 compared with CDK2; in order to understand the inhibitory mechanism of TEI-I01800, the cyclin-dependent kinase 2 (CDK2) complex structure with TEI-I01800 was determined at 2.0 Å resolution. Interestingly, the Gly-rich loop of CDK2 formed a β-sheet that was different from that of MK2. In MK2, TEI-I01800 changed the secondary structure of the Gly-rich loop from a β-sheet to an α-helix by collision between Leu70 and a p-ethoxyphenyl group at the 7-position and bound to MK2. However, for CDK2, TEI-I01800 bound to CDK2 without this structural change and lost the interaction with the substituent at the 7-position. In summary, the results of this study suggest that the reason for the selectivity of TEI-I01800 is the favourable conformation of TEI-I01800 itself, making it suitable for binding to the α-form MK2.

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

International Nuclear Information System (INIS)

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

2008-01-01

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

Budesonide Inhibits Intracellular Infection with Non-Typeable Haemophilus influenzae Despite Its Anti-Inflammatory Effects in Respiratory Cells and Human Lung Tissue: A Role for p38 MAP Kinase.

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Wagner, Christopher; Goldmann, Torsten; Rohmann, Kristina; Rupp, Jan; Marwitz, Sebastian; Rotta Detto Loria, Johannes; Limmer, Stefan; Zabel, Peter; Dalhoff, Klaus; Drömann, Daniel

2015-01-01

Inhaled corticosteroids (ICS) are widely used in the treatment of obstructive lung diseases. Recent data suggest a higher pneumonia risk in chronic obstructive pulmonary disease (COPD) patients treated with ICS. Since non-typeable Haemophilus influenzae (NTHi) is the most common pathogen associated with acute exacerbations of COPD, we investigated the effects of budesonide (BUD) on NTHi-induced inflammation and invasive infection. The alveolar epithelial cell line A549 and specimens of human lung tissue (HLT) were used in our experiments. Intracellular infection was determined by a lysis/culture assay of infected cells. Activated p38 mitogen-associated protein kinase (MAPK) was assessed using Western blotting and immunohistochemistry, expression of toll-like receptor 2 (TLR2) was determined by PCR, and CXCL-8 levels were measured using ELISA. Immunohistochemistry was used for detection of CXCL-8, platelet-activating factor receptor (PAF-R) and NTHi. BUD significantly reduced CXCL-8 secretion in A549 cells and lung tissue infected with NTHi. Furthermore, BUD decreased the expression of PAF-R in HLT and A549 cells. In A549 cells and HLT, BUD inhibited intracellular infection and - synergistically with NTHi - increased the expression of TLR2 (in A549 cells). TLR2 stimulation did not influence the intracellular infection of A549 cells, but p38 MAPK inhibition resulted in a significant reduction of infection. The present study adds new insights into the effects of glucocorticoids on pulmonary host defence after NTHi infection. Although the inflammatory response to infection is suppressed by BUD, interestingly, the intracellular infection is also inhibited. This effect seems to depend on the inhibition of p38 MAPK - a key enzyme in many pro-inflammatory pathways - as well as of PAF-R expression. © 2015 S. Karger AG, Basel.

Differential roles of MAPK-Erk1/2 and MAPK-p38 in insulin or insulin-like growth factor-I (IGF-I) signaling pathways for progesterone production in human ovarian cells.

Science.gov (United States)

Seto-Young, D; Avtanski, D; Varadinova, M; Park, A; Suwandhi, P; Leiser, A; Parikh, G; Poretsky, L

2011-06-01

Insulin and insulin like-growth factor-I (IGF-I) participate in the regulation of ovarian steroidogenesis. In insulin resistant states ovaries remain sensitive to insulin because insulin can activate alternative signaling pathways, such as phosphatidylinositol-3-kinase (PI-3 kinase) and mitogen-activated protein-kinase (MAPK) pathways, as well as insulin receptors and type 1 IGF receptors. We investigated the roles of MAPK-Erk1/2 and MAPK-p38 in insulin and IGF-I signaling pathways for progesterone production in human ovarian cells. Human ovarian cells were cultured in tissue culture medium in the presence of varying concentrations of insulin or IGF-I, with or without PD98059, a specific MAPK-Erk1/2 inhibitor, with or without SB203580, a specific MAPK-p38 inhibitor or with or without a specific PI-3-kinase inhibitor LY294002. Progesterone concentrations were measured using radioimmunoassay. PD98059 alone stimulated progesterone production in a dose-dependent manner by up to 65% (pprogesterone production by 13-18% (pprogesterone production by 17-20% (pprogesterone production by 20-30% (pprogesterone production by 40-60% (pprogesterone synthesis while SB203580 abolished insulin-induced progesterone production. Either PD98059 or SB203580 abolished IGF-I-induced progesterone production. Both MAPK-Erk1/2 and MAPK-p38 participate in IGF-I-induced signaling pathways for progesterone production, while insulin-induced progesterone production requires MAPK-p38, but not MAPK-Erk1/2. These studies provide further evidence for divergence of insulin and IGF-I signaling pathways for human ovarian cell steroidogenesis. © Georg Thieme Verlag KG Stuttgart · New York.

ROS detoxification and proinflammatory cytokines are linked by p38 MAPK signaling in a model of mature astrocyte activation.

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Adrian Nahirnyj

Full Text Available Astrocytes are the most abundant glial cell in the retinal nerve fiber layer (NFL and optic nerve head (ONH, and perform essential roles in maintaining retinal ganglion cell (RGC detoxification and homeostasis. Mature astrocytes are relatively quiescent, but rapidly undergo a phenotypic switch in response to insult, characterized by upregulation of intermediate filament proteins, loss of glutamate buffering, secretion of pro-inflammatory cytokines, and increased antioxidant production. These changes result in both positive and negative influences on RGCs. However, the mechanism regulating these responses is still unclear, and pharmacologic strategies to modulate select aspects of this switch have not been thoroughly explored. Here we describe a system for rapid culture of mature astrocytes from the adult rat retina that remain relatively quiescent, but respond robustly when challenged with oxidative damage, a key pathogenic stress associated with inner retinal injury. When primary astrocytes were exposed to reactive oxygen species (ROS we consistently observed characteristic changes in activation markers, along with increased expression of detoxifying genes, and secretion of proinflammatory cytokines. This in vitro model was then used for a pilot chemical screen to target specific aspects of this switch. Increased activity of p38α and β Mitogen Activated Protein Kinases (MAPKs were identified as a necessary signal regulating expression of MnSOD, and heme oxygenase 1 (HO-1, with consequent changes in ROS-mediated injury. Additionally, multiplex cytokine profiling detected p38 MAPK-dependent secretion of IL-6, MCP-1, and MIP-2α, which are proinflammatory signals recently implicated in damage to the inner retina. These data provide a mechanism to link increased oxidative stress to proinflammatory signaling by astrocytes, and establish this assay as a useful model to further dissect factors regulating the reactive switch.

Convergence of PASTA kinase and two-component signaling in response to cell wall stress in Enterococcus faecalis.

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Kellogg, Stephanie L; Kristich, Christopher J

2018-04-09

Two common signal transduction mechanisms used by bacteria to sense and respond to changing environments are two-component systems (TCSs) and eukaryotic-like Ser/Thr kinases and phosphatases (eSTK/Ps). Enterococcus faecalis is a Gram-positive bacterium and serious opportunistic pathogen that relies on both a TCS and an eSTK/P pathway for intrinsic resistance to cell wall-targeting antibiotics. The TCS consists of a histidine kinase (CroS) and response regulator (CroR) that become activated upon exposure of cells to cell wall-targeting antibiotics, leading to modulation of gene expression. The eSTK/P pathway consists of a transmembrane kinase (IreK) and its cognate phosphatase (IreP), which act antagonistically to mediate antibiotic resistance through an unknown mechanism. Because both CroS/R and IreK/P contribute to enterococcal resistance towards cell wall-targeting antibiotics, we hypothesized these signaling systems are intertwined. To test this hypothesis, we analyzed CroR phosphorylation and CroS/R-dependent gene expression to probe the influence of IreK and IreP on CroS/R signaling. In addition, we analyzed the phosphorylation state of CroS which revealed IreK-dependent phosphorylation of a Thr residue important for CroS function. Our results are consistent with a model in which IreK positively influences CroR-dependent gene expression through phosphorylation of CroS to promote antimicrobial resistance in E. faecalis Importance Two-component signaling systems (TCSs) and eukaryotic-like Ser/Thr kinases (eSTKs) are used by bacteria to sense and adapt to changing environments. Understanding how these pathways are regulated to promote bacterial survival is critical for a more complete understanding of bacterial stress responses and physiology. The opportunistic pathogen Enterococcus faecalis relies on both a TCS (CroS/R) and an eSTK (IreK) for intrinsic resistance to cell wall-targeting antibiotics. We probed the relationship between CroS/R and IreK, revealing

p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

Science.gov (United States)

Ettelaie, Camille; Elkeeb, Azza M; Maraveyas, Anthony; Collier, Mary Elizabeth W

2013-03-01

We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles. Copyright © 2012 Elsevier B.V. All rights reserved.

Deficiency of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 accelerates atherogenesis in apolipoprotein E-deficient mice

International Nuclear Information System (INIS)

Akyuerek, Levent M.; Boehm, Manfred; Olive, Michelle; Zhou, Alex-Xianghua; San, Hong; Nabel, Elizabeth G.

2010-01-01

Cyclin-dependent kinase inhibitors, p21 Cip1 and p27 Kip1 , are upregulated during vascular cell proliferation and negatively regulate growth of vascular cells. We hypothesized that absence of either p21 Cip1 or p27 Kip1 in apolipoprotein E (apoE)-deficiency may increase atherosclerotic plaque formation. Compared to apoE -/- aortae, both apoE -/- /p21 -/- and apoE -/- /p27 -/- aortae exhibited significantly more atherosclerotic plaque following a high-cholesterol regimen. This increase was particularly observed in the abdominal aortic regions. Deficiency of p27 Kip1 accelerated plaque formation significantly more than p21 -/- in apoE -/- mice. This increased plaque formation was in parallel with increased intima/media area ratios. Deficiency of p21 Cip1 and p27 Kip1 accelerates atherogenesis in apoE -/- mice. These findings have significant implications for our understanding of the molecular basis of atherosclerosis associated with excessive proliferation of vascular cells.

MaHog1, a Hog1-type mitogen-activated protein kinase gene, contributes to stress tolerance and virulence of the entomopathogenic fungus Metarhizium acridum.

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Jin, Kai; Ming, Yue; Xia, Yu Xian

2012-12-01

Fungal biocontrol agents have great potential in integrated pest management. However, poor efficacy and sensitivity to various adverse factors have hampered their wide application. In eukaryotic cells, Hog1 kinase plays a critical role in stress responses. In this study, MaHog1 (GenBank accession no. EFY85878), encoding a member of the Hog1/Sty1/p38 mitogen-activated protein kinase family in Metarhizium (Me.) acridum, was identified. Targeted gene disruption was used to analyse the role of MaHog1 in virulence and tolerance of adverse factors. Mutants with MaHog1 depletion showed increased sensitivity to high osmotic stress, high temperature and oxidative stress, and exhibited remarkable resistance to cell wall-disturbing agents. These results suggest that Hog1 kinase has a conserved function in regulating multistress responses among fungi, and that MaHog1 might influence cell wall biogenesis in Me. acridum. Bioassays conducted with topical inoculation and intrahaemocoel injection revealed that MaHog1 is required for both penetration and postpenetration development of Me. acridum. MaHog1 disruption resulted in a significant reduction in virulence, likely due to the combination of a decrease in conidial germination, a reduction in appressorium formation and a decline in growth rate in insect haemolymph, which might be caused by impairing fungal tolerance of various stresses during infection.

Clinical significance of cyclin-dependent kinase inhibitor p27Kip1 expression and proliferation in non-Hodgkin's lymphoma

DEFF Research Database (Denmark)

Møller, Michael Boe; Skjødt, Karsten; Mortensen, Leif Spange

1999-01-01

The cyclin-dependent kinase inhibitor p27Kip1 is a negative cell cycle regulator linking extracellular growth-regulatory signals to the cell cycle machinery in G1. We investigated the pattern and prognostic value of p27Kip1 expression in a population-based group of 203 non-Hodgkin's lymphoma (NHL...... between p27Kip1 and Ki-67 expression. Low expression of p27Kip1, defined as nuclear p27Kip1 expression in lymphomas behaved differently as those with low p27Kip1...... expression tended to do better. Likewise, a high proliferation rate (Ki-67 >40%) was associated with poor survival in indolent and aggressive lymphomas. Multivariate analysis using the proportional hazards model showed that only p27Kip1, and not Ki-67, maintained independent prognostic significance...

The structure of arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

KAUST Repository

Yunta, Cristina; Martí nez-Ripoll, Martí n; Zhu, Jian-Kang; Albert, Armando

2011-01-01

SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. © 2011 Elsevier Ltd. All rights reserved.

The structure of arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

KAUST Repository

Yunta, Cristina

2011-11-01

SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. © 2011 Elsevier Ltd. All rights reserved.

p38 phosphorylation in medullary microglia mediates ectopic orofacial inflammatory pain in rats.

Science.gov (United States)

Kiyomoto, Masaaki; Shinoda, Masamichi; Honda, Kuniya; Nakaya, Yuka; Dezawa, Ko; Katagiri, Ayano; Kamakura, Satoshi; Inoue, Tomio; Iwata, Koichi

2015-08-12

Orofacial inflammatory pain is likely to accompany referred pain in uninflamed orofacial structures. The ectopic pain precludes precise diagnosis and makes treatment problematic, because the underlying mechanism is not well understood. Using the established ectopic orofacial pain model induced by complete Freund's adjuvant (CFA) injection into trapezius muscle, we analyzed the possible role of p38 phosphorylation in activated microglia in ectopic orofacial pain. Mechanical allodynia in the lateral facial skin was induced following trapezius muscle inflammation, which accompanied microglial activation with p38 phosphorylation and hyperexcitability of wide dynamic range (WDR) neurons in the trigeminal spinal subnucleus caudalis (Vc). Intra-cisterna successive administration of a p38 mitogen-activated protein kinase selective inhibitor, SB203580, suppressed microglial activation and its phosphorylation of p38. Moreover, SB203580 administration completely suppressed mechanical allodynia in the lateral facial skin and enhanced WDR neuronal excitability in Vc. Microglial interleukin-1β over-expression in Vc was induced by trapezius muscle inflammation, which was significantly suppressed by SB203580 administration. These findings indicate that microglia, activated via p38 phosphorylation, play a pivotal role in WDR neuronal hyperexcitability, which accounts for the mechanical hypersensitivity in the lateral facial skin associated with trapezius muscle inflammation.

Ribosomal stress induces L11- and p53-dependent apoptosis in mouse pluripotent stem cells.

Science.gov (United States)

Morgado-Palacin, Lucia; Llanos, Susana; Serrano, Manuel

2012-02-01

Ribosome biogenesis is the most demanding energetic process in proliferating cells and it is emerging as a critical sensor of cellular homeostasis. Upon disturbance of ribosome biogenesis, specific free ribosomal proteins, most notably L11, bind and inhibit Mdm2, resulting in activation of the tumor suppressor p53. This pathway has been characterized in somatic and cancer cells, but its function in embryonic pluripotent cells has remained unexplored. Here, we show that treatment with low doses of Actinomycin D or depletion of ribosomal protein L37, two well-established inducers of ribosomal stress, activate p53 in an L11-dependent manner in mouse embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). Activation of p53 results in transcriptional induction of p53 targets, including p21, Mdm2, Pidd, Puma, Noxa and Bax. Finally, ribosomal stress elicits L11- and p53-dependent apoptosis in ESCs/iPSCs. These results extend to pluripotent cells the functionality of the ribosomal stress pathway and we speculate that this could be a relevant cellular checkpoint during early embryogenesis.

Structure-based design, synthesis and crystallization of 2-arylquinazolines as lipid pocket ligands of p38α MAPK.

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Mike Bührmann

Full Text Available In protein kinase research, identifying and addressing small molecule binding sites other than the highly conserved ATP-pocket are of intense interest because this line of investigation extends our understanding of kinase function beyond the catalytic phosphotransfer. Such alternative binding sites may be involved in altering the activation state through subtle conformational changes, control cellular enzyme localization, or in mediating and disrupting protein-protein interactions. Small organic molecules that target these less conserved regions might serve as tools for chemical biology research and to probe alternative strategies in targeting protein kinases in disease settings. Here, we present the structure-based design and synthesis of a focused library of 2-arylquinazoline derivatives to target the lipophilic C-terminal binding pocket in p38α MAPK, for which a clear biological function has yet to be identified. The interactions of the ligands with p38α MAPK was analyzed by SPR measurements and validated by protein X-ray crystallography.

3,3'-Diindolylmethane is a novel mitochondrial H(+)-ATP synthase inhibitor that can induce p21(Cip1/Waf1) expression by induction of oxidative stress in human breast cancer cells.

Science.gov (United States)

Gong, Yixuan; Sohn, Heesook; Xue, Ling; Firestone, Gary L; Bjeldanes, Leonard F

2006-05-01

Epidemiologic evidence suggests that high dietary intake of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis in multiple organs. 3,3'-Diindolylmethane, one of the active products derived from Brassica vegetables, is a promising antitumor agent. Previous studies in our laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in human breast cancer MCF-7 cells by a mechanism that included increased expression of p21. In the present study, the upstream events leading to p21 overexpression were further investigated. We show for the first time that 3,3'-diindolylmethane is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20 micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of mitochondrial inner membrane, decreased cellular ATP level, and significantly stimulated mitochondrial reactive oxygen species (ROS) production. ROS production, in turn, led to the activation of stress-activated pathways involving p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580 and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase (JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced activation of p38 and JNK and induction of p21, indicating that oxidative stress is the major trigger of these events. To further support the role of ROS in 3,3'-diindolylmethane-induced p21 overexpression, we showed that 3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane did not stimulate ROS production. Thus, we have established the critical role of enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21 up-regulation in human breast cancer cells.

JS-III-49, a hydroquinone derivative, exerts anti-inflammatory activity by targeting Akt and p38.

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Yi, Young-Su; Kim, Mi-Yeon; Cho, Jae Youl

2017-05-01

Since previous studies have reported that hydroquinone (HQ) exerted immunosuppressive and anti-inflammatory activity, various HQ derivatives have been synthesized and their biological activities investigated. In this study, we explored the anti-inflammatory activity of JS-III-49, a novel HQ derivative, in macrophage-mediated inflammatory responses. JS-III-49 suppressed the production of the inflammatory mediators nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) and down-regulated the mRNA expression of the inflammatory enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as the expression of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1b without cytotoxicity in LPS-stimulated RAW264.7 cells. JS-III-49 inhibited nuclear translocation of the NF-kB transcription factors p65 and p50 by directly targeting Akt, an upstream kinase of the NF-kB pathway, in LPS-stimulated RAW264.7 cells. However, JS-III-49 did not directly inhibit the kinase activities of Src and Syk, which are upstream kinases of Akt, in LPS-stimulated RAW264.7 cells. Moreover, JS-III-49 suppressed the nuclear translocation of c-Fos, one of the components of AP-1, by specifically targeting p38, an upstream mitogen-activated protein kinase (MAPK) in the AP-1 pathway in LPS-stimulated RAW264.7 cells. These results suggest that JS-III-49 plays an anti-inflammatory role in LPS-stimulated macrophages by targeting Akt and p38 in the NF-kB and AP-1 pathways, respectively.

Aconitine-induced Ca2+ overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats

International Nuclear Information System (INIS)

Sun, Gui-bo; Sun, Hong; Meng, Xiang-bao; Hu, Jin; Zhang, Qiang; Liu, Bo; Wang, Min; Xu, Hui-bo; Sun, Xiao-bo

2014-01-01

Aconitine is a major bioactive diterpenoid alkaloid with high content derived from herbal aconitum plants. Emerging evidence indicates that voltage-dependent Na + channels have pivotal roles in the cardiotoxicity of aconitine. However, no reports are available on the role of Ca 2+ in aconitine poisoning. In this study, we explored the importance of pathological Ca 2+ signaling in aconitine poisoning in vitro and in vivo. We found that Ca 2+ overload lead to accelerated beating rhythm in adult rat ventricular myocytes and caused arrhythmia in conscious freely moving rats. To investigate effects of aconitine on myocardial injury, we performed cytotoxicity assay in neonatal rat ventricular myocytes (NRVMs), as well as measured lactate dehydrogenase level in the culture medium of NRVMs and activities of serum cardiac enzymes in rats. The results showed that aconitine resulted in myocardial injury and reduced NRVMs viability dose-dependently. To confirm the pro-apoptotic effects, we performed flow cytometric detection, cardiac histology, transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The results showed that aconitine stimulated apoptosis time-dependently. The expression analysis of Ca 2+ handling proteins demonstrated that aconitine promoted Ca 2+ overload through the expression regulation of Ca 2+ handling proteins. The expression analysis of apoptosis-related proteins revealed that pro-apoptotic protein expression was upregulated, and anti-apoptotic protein BCL-2 expression was downregulated. Furthermore, increased phosphorylation of MAPK family members, especially the P-P38/P38 ratio was found in cardiac tissues. Hence, our results suggest that aconitine significantly aggravates Ca 2+ overload and causes arrhythmia and finally promotes apoptotic development via phosphorylation of P38 mitogen-activated protein kinase. - Highlights: • Aconitine-induced Ca 2+ overload causes arrhythmia in rats

Inhibition of stress-activated MAP kinases induces clinical improvement in moderate to severe Crohn's disease

NARCIS (Netherlands)

Hommes, Daan; van den Blink, Bernt; Plasse, Terry; Bartelsman, Joep; Xu, Cuiping; Macpherson, Bret; Tytgat, Guido; Peppelenbosch, Mailkel; van Deventer, Sander

2002-01-01

Background & Aims: We investigated if inhibition of mitogen-activated protein kinases (MAPKs) was beneficial in Crohn's disease. Methods: Inhibition of JNK and p38 MAPK activation with CNI-1493, a guanylhydrazone, was tested in vitro. Twelve patients with severe Crohn's disease (mean baseline, CDAI

Curcumin I mediates neuroprotective effect through attenuation of quinoprotein formation, p-p38 MAPK expression, and caspase-3 activation in 6-hydroxydopamine treated SH-SY5Y cells.

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Meesarapee, Benjawan; Thampithak, Anusorn; Jaisin, Yamaratee; Sanvarinda, Pimtip; Suksamrarn, Apichart; Tuchinda, Patoomratana; Morales, Noppawan Phumala; Sanvarinda, Yupin

2014-04-01

6-Hydroxydopamine (6-OHDA) selectively enters dopaminergic neurons and undergoes auto-oxidation resulting in the generation of reactive oxygen species and dopamine quinones, subsequently leading to apoptosis. This mechanism mimics the pathogenesis of Parkinson's disease and has been used to induce experimental Parkinsonism in both in vitro and in vivo systems. In this study, we investigated the effects of curcumin I (diferuloylmethane) purified from Curcuma longa on quinoprotein production, phosphorylation of p38 MAPK (p-p38), and caspase-3 activation in 6-OHDA-treated SH-SY5Y dopaminergic cells. Pretreatment of SH-SY5Y with curcumin I at concentrations of 1, 5, 10, and 20 μM, significantly decreased the formation of quinoprotein and reduced the levels of p-p38 and cleaved caspase-3 in a dose-dependent manner. Moreover, the levels of the dopaminergic neuron marker, phospho-tyrosine hydroxylase (p-TH), were also dose-dependently increased upon treatment with curcumin I. Our results clearly demonstrated that curcumin I protects neurons against oxidative damage, as shown by attenuation of p-p38 expression, caspase-3-activation, and toxic quinoprotein formation, together with the restoration of p-TH levels. This study provides evidence for the therapeutic potential of curcumin I in the chemoprevention of oxidative stress-related neurodegeneration. Copyright © 2013 John Wiley & Sons, Ltd.

Recent Advances in the Inhibition of p38 MAPK as a Potential Strategy for the Treatment of Alzheimer's Disease.

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Lee, Jong Kil; Kim, Nam-Jung

2017-08-02

P38 mitogen-activated protein kinase (MAPK) is a crucial target for chronic inflammatory diseases. Alzheimer's disease (AD) is characterized by the presence of amyloid plaques and neurofibrillary tangles in the brain, as well as neurodegeneration, and there is no known cure. Recent studies on the underlying biology of AD in cellular and animal models have indicated that p38 MAPK is capable of orchestrating diverse events related to AD, such as tau phosphorylation, neurotoxicity, neuroinflammation and synaptic dysfunction. Thus, the inhibition of p38 MAPK is considered a promising strategy for the treatment of AD. In this review, we summarize recent advances in the targeting of p38 MAPK as a potential strategy for the treatment of AD and envision possibilities of p38 MAPK inhibitors as a fundamental therapeutics for AD.

Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases.

Science.gov (United States)

Zhu, Guozhi; Fujii, Koichi; Belkina, Natalya; Liu, Yin; James, Michael; Herrero, Juan; Shaw, Stephen

2005-03-18

To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.

WNK1 and p38-MAPK distribution in ionocytes and accessory cells of euryhaline teleost fish implies ionoregulatory function

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W. S. Marshall

2017-07-01

Full Text Available Ionocytes of euryhaline teleost fish secrete NaCl, under regulation by serine and threonine kinases, including with-no-lysine kinase (WNK1 and p38 mitogen-activated protein kinase (MAPK. Mummichogs (Fundulus heteroclitus L. were acclimated to freshwater (FW, full strength seawater (SW and hypersaline conditions (2SW. Immunocytochemistry of ionocytes in opercular epithelia of fish acclimated to SW and 2SW revealed that WNK1-anti-pT58 phosphoantibody localized strongly to accessory cells and was present in the cytosol of ionocytes, close to cystic fibrosis transmembrane conductance regulator (CFTR in the apical membrane and the sodium potassium 2 chloride cotransporter (NKCC in the basolateral membrane. In FW acclimated fish, WNK1 localized to a sub-apical zone, did not colocalize with apical membrane-located sodium chloride cotransporter (NCC, and typically was present in one cell of paired ionocytes and in some single ionocytes. Forskolin treatment (10 μM, 30 min increased WNK1 immunofluorescence in SW ionocytes only, while hypertonicity had little effect, compared to controls. Anti-p38-MAPK antibody localized to the cytosolic compartment. The distribution of WNK1 and p38MAPK is consistent with a proximal position in regulatory cascades, rather than directly affecting transporters. The strong staining of accessory cells by WNK1 phosphoantibody infers an osmoregulatory function for WNK.

Hydrogen sulfide protects against chemical hypoxia-induced injury by inhibiting ROS-activated ERK1/2 and p38MAPK signaling pathways in PC12 cells.

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Aiping Lan

Full Text Available Hydrogen sulfide (H(2S has been proposed as a novel neuromodulator and neuroprotective agent. Cobalt chloride (CoCl(2 is a well-known hypoxia mimetic agent. We have demonstrated that H(2S protects against CoCl(2-induced injuries in PC12 cells. However, whether the members of mitogen-activated protein kinases (MAPK, in particular, extracellular signal-regulated kinase1/2(ERK1/2 and p38MAPK are involved in the neuroprotection of H(2S against chemical hypoxia-induced injuries of PC12 cells is not understood. We observed that CoCl(2 induced expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α, decreased cystathionine-β synthase (CBS, a synthase of H(2S expression, and increased generation of reactive oxygen species (ROS, leading to injuries of the cells, evidenced by decrease in cell viability, dissipation of mitochondrial membrane potential (MMP , caspase-3 activation and apoptosis, which were attenuated by pretreatment with NaHS (a donor of H(2S or N-acetyl-L cystein (NAC, a ROS scavenger. CoCl(2 rapidly activated ERK1/2, p38MAPK and C-Jun N-terminal kinase (JNK. Inhibition of ERK1/2 or p38MAPK or JNK with kinase inhibitors (U0126 or SB203580 or SP600125, respectively or genetic silencing of ERK1/2 or p38MAPK by RNAi (Si-ERK1/2 or Si-p38MAPK significantly prevented CoCl(2-induced injuries. Pretreatment with NaHS or NAC inhibited not only CoCl(2-induced ROS production, but also phosphorylation of ERK1/2 and p38MAPK. Thus, we demonstrated that a concurrent activation of ERK1/2, p38MAPK and JNK participates in CoCl(2-induced injuries and that H(2S protects PC12 cells against chemical hypoxia-induced injuries by inhibition of ROS-activated ERK1/2 and p38MAPK pathways. Our results suggest that inhibitors of ERK1/2, p38MAPK and JNK or antioxidants may be useful for preventing and treating hypoxia-induced neuronal injury.

Limb Remote Ischemic Postconditioning Reduces Ischemia-Reperfusion Injury by Inhibiting NADPH Oxidase Activation and MyD88-TRAF6-P38MAP-Kinase Pathway of Neutrophils

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Gangling Chen

2016-11-01

Full Text Available Limb remote ischemic postconditioning (LRIP has been confirmed to reduce the ischemia-reperfusion injury but its mechanisms are still not clear. This study clarified the mechanism of LRIP based on the nicotinamide-adenine dinucleotide phosphate (NADPH oxidase and Myeloid differentiation factor 88 (MyD88-Tumor necrosis factor (TNF receptor-associated factor 6 (TRAF6-P38 pathway of neutrophils. Rat middle cerebral artery occlusion (MCAO model was used in this study. Ischemia-reperfusion injury was carried out by MCAO 1.5 h followed by 24 h reperfusion. LRIP operation was performed to the left femoral artery at 0, 1 or 3 h after reperfusion. Behavioral testing, including postural reflex test, vibrissae-elicited forelimb placing test and tail hang test, showed that LRIP operated at 0 h of reperfusion could significantly ameliorate these behavioral scores. Pathological examinations, infarct size, Myeloperoxidase (MPO activity showed that LRIP operated at 0 h of reperfusion could significantly ameliorate the pathological scores, reduce the infarct size and MPO activity in the brain and increase the MPO activity in the left leg. By using Neutrophil counting, immunofluorescence and real-time PCR techniques, we found that LRIP operated at 0 h of reperfusion could reduce neutrophil counts in the peripheral blood and downregulate the activation of neutrophil in the peripheral blood and rat brain. Western blots revealed that MyD88, TRAF6, p38 mitogen-activated protein kinase (p38-MAPK in neutrophils and the phosphorylation of p47phox (Ser 304 and Ser 345 in neutrophil could be downregulated by LRIP. Our study suggests that LRIP inhibits the number and activation of neutrophils in the rat brain and peripheral blood linked to down-regulating the activation of NADPH oxidase in neutrophils by MyD88/TRAF6/p38-MAPK pathway.

Genome-wide Identification and Expression Analysis of Calcium-dependent Protein Kinase and Its Closely Related Kinase Genes in Capsicum annuum

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hanyang ecai

2015-09-01

Full Text Available As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs play important roles in regulating the downstream components of calcium signaling, which are ubiquitously involved in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a comprehensive analysis of genes encoding pepper CDPKs and CDPK-related protein kinases (CRKs was performed, and 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CaCPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the Capsicum annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and eight CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families.