Mitogen-activated Protein Kinase In Response Research Articles

Harnessing the role of mitogen-activated protein kinases against abiotic stresses in plants.

Crop plants are vulnerable to various biotic and abiotic stresses, whereas plants tend to retain their physiological mechanisms by evolving cellular regulation. To mitigate the adverse effects of abiotic stresses, many defense mechanisms are induced in plants. One of these mechanisms is the mitogen-activated protein kinase (MAPK) cascade, a signaling pathway used in the transduction of extracellular stimuli into intercellular responses. This stress signaling pathway is activated by a series of responses involving MAPKKKs→MAPKKs→MAPKs, consisting of interacting proteins, and their functions depend on the collaboration and activation of one another by phosphorylation. These proteins are key regulators of MAPK in various crop plants under abiotic stress conditions and also related to hormonal responses. It is revealed that in response to stress signaling, MAPKs are characterized as multigenic families and elaborate the specific stimuli transformation as well as the antioxidant regulation system. This pathway is directed by the framework of proteins and stopping domains confer the related associates with unique structure and functions. Early studies of plant MAPKs focused on their functions in model plants. Based on the results of whole-genome sequencing, many MAPKs have been identified in plants, such as Arbodiposis, tomato, potato, alfalfa, poplar, rice, wheat, maize, and apple. In this review, we summarized the recent work on MAPK response to abiotic stress and the classification of MAPK cascade in crop plants. Moreover, we highlighted the modern research methodologies such as transcriptomics, proteomics, CRISPR/Cas technology, and epigenetic studies, which proposed, identified, and characterized the novel genes associated with MAPKs and their role in plants under abiotic stress conditions. In-silico-based identification of novel MAPK genes also facilitates future research on MAPK cascade identification and function in crop plants under various stress conditions.

Gene Expression Profiling Elucidates Cellular Responses to NCX4040 in Human Ovarian Tumor Cells: Implications in the Mechanisms of Action of NCX4040.

The nitric oxide donor, NCX4040 is a non-steroidal anti-inflammatory-NO donor and has been shown to be extremely cytotoxic to a number of human tumors, including ovarian tumors cells. We have found that NCX4040 is cytotoxic against both OVCAR-8 and its adriamycin-selected OVCAR-8 variant (NCI/ADR-RES) tumor cell lines. While the mechanism of action of NCX4040 is not entirely clear, we as well as others have shown that NCX4040 generates reactive oxygen species (ROS) and induces DNA damage in tumor cells. Recently, we have reported that NCX4040 treatment resulted in a significant depletion of cellular glutathione, and formation of both reactive oxygen and nitrogen species (ROS/RNS), resulting in oxidative stress in these tumor cells. Furthermore, our results indicated that more ROS/RNS were generated in OVCAR-8 cells than in NCI/ADR-RES cells due to increased activities of superoxide dismutase (SOD), glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. Further studies suggested that NCX4040-induced cell death may be mediated by peroxynitrite formed from NCX4040 in cells. In this study we used microarray analysis following NCX4040 treatment of both OVCAR-8 and its ADR-resistant variant to identify various molecular pathways involved in NCX4040-induced cell death. Here, we report that NCX4040 treatment resulted in the differential induction of oxidative stress genes, inflammatory response genes (TNF, IL-1, IL-6 and COX2), DNA damage response and MAP kinase response genes. A mechanism of tumor cell death is proposed based on our findings where oxidative stress is induced by NCX4040 from simultaneous induction of NOX4, TNF-α and CHAC1 in tumor cell death.

MAPK Cascades and Transcriptional Factors: Regulation of Heavy Metal Tolerance in Plants.

In nature, heavy metal (HM) stress is one of the most destructive abiotic stresses for plants. Heavy metals produce toxicity by targeting key molecules and important processes in plant cells. The mitogen-activated protein kinase (MAPK) cascade transfers the signals perceived by cell membrane surface receptors to cells through phosphorylation and dephosphorylation and targets various effector proteins or transcriptional factors so as to result in the stress response. Signal molecules such as plant hormones, reactive oxygen species (ROS), and nitric oxide (NO) can activate the MAPK cascade through differentially expressed genes, the activation of the antioxidant system and synergistic crosstalk between different signal molecules in order to regulate plant responses to HMs. Transcriptional factors, located downstream of MAPK, are key factors in regulating plant responses to heavy metals and improving plant heavy metal tolerance and accumulation. Thus, understanding how HMs activate the expression of the genes related to the MAPK cascade pathway and then phosphorylate those transcriptional factors may allow us to develop a regulation network to increase our knowledge of HMs tolerance and accumulation. This review highlighted MAPK pathway activation and responses under HMs and mainly focused on the specificity of MAPK activation mediated by ROS, NO and plant hormones. Here, we also described the signaling pathways and their interactions under heavy metal stresses. Moreover, the process of MAPK phosphorylation and the response of downstream transcriptional factors exhibited the importance of regulating targets. It was conducive to analyzing the molecular mechanisms underlying heavy metal accumulation and tolerance.

Visualizing cellular heterogeneity by quantifying the dynamics of MAPK activity in live mammalian cells with synthetic fluorescent biosensors

Mitogen-Activated Protein Kinases (MAPKs) control a wide array of cellular functions by transducing extracellular information into defined biological responses. In order to understand how these pathways are regulated, dynamic single cell measurements are highly needed. Fluorescence microscopy is well suited to perform these measurements. However, more dynamic and sensitive biosensors that allow the quantification of signaling activity in living mammalian cells are required. We have engineered a synthetic fluorescent substrate for human MAPKs (ERK, JNK and p38) that relocates from the nucleus to the cytoplasm when phosphorylated by the kinases. We demonstrate that this reporter displays an improved response compared to other relocation biosensors. This assay allows to monitor the heterogeneity in the MAPK response in a population of isogenic cells, revealing pulses of ERK activity upon a physiological EGFR stimulation. We show applicability of this approach to the analysis of multiple cancer cell lines and primary cells as well as its application in vivo to developing tumors. Using this ERK biosensor, dynamic single cell measurements with high temporal resolution can be obtained. These MAPK reporters can be widely applied to the analysis of molecular mechanisms of MAPK signaling in healthy and diseased state, in cell culture assays or in vivo.

Expression of signal-transducing adaptor protein-1 attenuates experimental autoimmune hepatitis via down-regulating activation and homeostasis of invariant natural killer T cells.

ObjectiveSignal-transducing adaptor protein (STAP) family members function as adaptor molecules and are involved in several events during immune responses. Notably however, the biological functions of STAP-1 in other cells are not known. We aimed to investigate the functions of STAP-1 in invariant natural killer T (iNKT) cells and iNKT cell-dependent hepatitis.MethodsWe employed concanavalin A (Con A)-induced hepatitis and α-galactosylceramide (α-GalCer)-induced hepatitis mouse models, both are models of iNKT cell-dependent autoimmune hepatitis, and STAP-1 overexpressing 2E10 cells to investigate the role of STAP-1 in iNKT cell activation in vivo an in vitro, respectively.ResultsAfter Con A- or α-GalCer-injection, hepatocyte necrotic areas and plasma alanine aminotransferase elevation were more severe in STAP-1 knockout (S1KO) mice and milder in lymphocyte-specific STAP-1 transgenic (S1Tg) mice, as compared to wild-type (WT) mice. Two events that may be related to Con A-induced and/or α-GalCer-induced hepatitis were influenced by STAP-1 manipulation. One is that iNKT cell populations in the livers and spleens were increased in S1KO mice and were decreased in S1Tg mice. The other is that Con A-induced interleukin-4 and interferon-γ production was attenuated by STAP-1 overexpression. These effects of STAP-1 were confirmed using 2E10 cells overexpressing STAP-1 that showed impairment of interleukin-4 and interferon-γ production as well as phosphorylation of Akt and mitogen-activated protein kinases in response to Con A stimulation.ConclusionsThese results conclude that STAP-1 regulates iNKT cell maintenance/activation, and is involved in the pathogenesis of autoimmune hepatitis.

Expression Levels of the Immune-Related p38 Mitogen-Activated Protein Kinase Transcript in Response to Environmental Pollutants on Macrophthalmus japonicus Crab.

Environmental pollution in the aquatic environment poses a threat to the immune system of benthic organisms. The Macrophthalmus japonicus crab, which inhabits tidal flat sediments, is a marine invertebrate that provides nutrient and organic matter cycling as a means of purification. Here, we characterized the M. japonicus p38 mitogen-activated protein kinase (MAPK) gene, which plays key roles in the regulation of cellular immune and apoptosis responses. M. japonicus p38 MAPK displayed the characteristics of the conserved MAPK family with Thr-Gly-Tyr (TGY) motif and substrate-binding site Ala-Thr-Arg-Trp (ATRW). The amino acid sequence of the M. japonicus p38 MAPK showed a close phylogenetic relationship to Eriocheir sinensis MAPK14 and Scylla paramamosain p38 MAPK. The phylogenetic tree displayed two origins of p38 MAPK: crustacean and insect. The tissue distribution patterns showed the highest expression in the gills and hepatopancreas of M. japonicus crab. In addition, p38 MAPK expression in M. japonicus gills and hepatopancreas was evaluated after exposure to environmental pollutants such as perfluorooctane sulfonate (PFOS), irgarol, di(2-ethylhexyl) phthalate (DEHP), and bisphenol A (BPA). In the gills, p38 MAPK expression significantly increased after exposure to all concentrations of the chemicals on day 7. However, on day 1, there were increased p38 MAPK responses observed after PFOS and irgarol exposure, whereas decreased p38 MAPK responses were observed after DEHP and BPA exposure. The upregulation of p38 MAPK gene also significantly led to M. japonicus hepatopancreas being undertested in all environmental pollutants. The findings in this study supported that anti-stress responses against exposure to environmental pollutants were reflected in changes in expression levels in M. japonicus p38 MAPK signaling regulation as a cellular defense mechanism.

Histone Deacetylase Inhibitors to Overcome Resistance to Targeted and Immuno Therapy in Metastatic Melanoma.

Therapies that target oncogenes and immune checkpoint molecules constitute a major group of treatments for metastatic melanoma. A mutation in BRAF (BRAF V600E) affects various signaling pathways, including mitogen activated protein kinase (MAPK) and PI3K/AKT/mammalian target of rapamycin (mTOR) in melanoma. Target-specific agents, such as MAPK inhibitors improve progression-free survival. However, BRAFV600E mutant melanomas treated with BRAF kinase inhibitors develop resistance. Immune checkpoint molecules, such as programmed death-1 (PD-1) and programmed death ligand-1(PD-L1), induce immune evasion of cancer cells. MAPK inhibitor resistance results from the increased expression of PD-L1. Immune checkpoint inhibitors, such as anti-PD-L1 or anti-PD-1, are main players in immune therapies designed to target metastatic melanoma. However, melanoma patients show low response rate and resistance to these inhibitors develops within 6–8 months of treatment. Epigenetic reprogramming, such as DNA methylaion and histone modification, regulates the expression of genes involved in cellular proliferation, immune checkpoints and the response to anti-cancer drugs. Histone deacetylases (HDACs) remove acetyl groups from histone and non-histone proteins and act as transcriptional repressors. HDACs are often dysregulated in melanomas, and regulate MAPK signaling, cancer progression, and responses to various anti-cancer drugs. HDACs have been shown to regulate the expression of PD-1/PD-L1 and genes involved in immune evasion. These reports make HDACs ideal targets for the development of anti-melanoma therapeutics. We review the mechanisms of resistance to anti-melanoma therapies, including MAPK inhibitors and immune checkpoint inhibitors. We address the effects of HDAC inhibitors on the response to MAPK inhibitors and immune checkpoint inhibitors in melanoma. In addition, we discuss current progress in anti-melanoma therapies involving a combination of HDAC inhibitors, immune checkpoint inhibitors, and MAPK inhibitors.

Decoding patterns of macrophage TNF production to model NF-κB dynamics and cell-cell communication

Abstract Immune and stromal cells communicate through cytokines to initiate intracellular signaling, gene expression, and a multifaceted immune response. Cytokine signaling coordinates the dynamics of intracellular pathways that regulate inflammation. Immune and stromal cells both produce and respond to tumor necrosis factor-alpha (TNF), mediating inflammation and enabling communication between these compartments. The inflammatory role of TNF, activating the nuclear factor-κB (NF-κB) signaling and mitogen-activated protein kinase (MAPK) pathways, is well known. However, the effect of varying the time and amplitude patterns of TNF production in neighboring immune and stromal cells has not been systematically studied. Moreover, little is known about how time and amplitude patterns of an inflammatory signal input impact the nature of inflammatory responses. To study this relationship between production and response to TNF and its role in the dynamics of TNF-dependent NF-κB and MAPK signaling, we are using a systems immunology approach. Specifically, we are experimentally determining the patterns of TNF produced by human macrophages stimulated with heat-killed bacteria and characterizing the effect of these TNF stimulation patterns in the dynamics of NF-κB and MAPK responses in human cell lines. Additionally, we will use these experimental results to calibrate our deterministic 2 feedback with competitor (D2FC) mathematical model and model the dynamics of NF-κB signaling. Findings from this research will likely apply to other cytokine and signaling pathways, contributing to understanding the integration of time varying cytokine stimuli.

Downregulation of mitogen-activated protein kinases (MAPKs) in chronic ethanol-induced fatty liver

Mitogen-activated protein kinases (MAPKs) are versatile proteins that have been suggested to be involved in the regulation of lipid metabolism. This study was designed to investigate the responses of MAPK signaling to chronic ethanol exposure in vivo and in vitro, and try to explore its role in the pathogenesis of alcoholic fatty liver (AFL). Mice were fed with Lieber-Decarli liquid diet (5% ethanol, w/v) for 4 weeks to induce fatty liver, and the chronological changes of MAPK phosphorylation were measured using western blotting. We found that chronic ethanol feeding led to accumulation of triglyceride (TG), decreased phosphorylation of MAPKs, decreased protein level of peroxisomal proliferator activation receptor α (PPARα), and increased protein expression of cytochrome P4502E1 (CYP2E1) in mice liver. In vitro study showed that overexpression of CYP2E1 blunted the response of MAPKs to ethanol, and MAPK phosphatase 1 (MKP-1) knockdown by siRNA led to upregulation of PPARα protein level. Lastly, epidermal growth factor (EGF), a well-known MAPK activator, significantly suppressed chronic ethanol-induced hepatic fat accumulation and decline of PPARα expression in mice liver. Collectively, MAPK suppression, possibly due to the activation of hepatic CYP2E1, may be involved in chronic ethanol-induced hepatic steatosis.

The effect of a high fat diet in the regulation of MAP kinase in left ventricle fibrosis

IntroductionIt is well known that obesity contributes to the development of systemic inflammatory responses, which in turn may be involved in the process of interstitial fibrosis and left ventricular (LV) remodeling. Activation of proinflammatory factors such as transforming growth factor β (TGF‐β) can directly stimulate mitogen‐activated protein kinase (MAPK) p38 and JNK.PurposeThe aim of this study was to evaluate the level of TGF‐β, MAPK p38 and JNK in the LV in Sprague Dawley (SPRD) rats maintained on a high fat diet (HFD).MethodsThe SPRD rats were maintained on a normal fat diet (NFD) or on a HFD from 4 weeks of age for 12 weeks (NFD 16‐week‐old rats, NFD 16‐wk; or HFD 16‐week‐old rats, HFD 16‐wk) or for 16 weeks (NFD 20‐week‐old rats, NFD 20‐wk; or HFD 20‐week‐old rats, HFD 20‐wk). At the end of the experiment, blood and LV were collected from all rats for further analysis (Real‐Time PCR, histopathological and immunohistochemical analysis). Consent was obtained from The Local Animal Research Ethics Committee (33/2011).ResultsThe LV fibrosis area in relation to the entire LV area was significantly higher in the HFD 16‐wk rats and the HFD 20‐wk rats compared with the NFD 16‐wk rats and the NFD 20‐wk rats, respectively. TGF‐β mRNA expression did not differ between the study groups of rats. However, p38 MAPK mRNA expression and c‐jun mRNA expression were significantly lower in the HFD 20‐wk rats than in the HFD 16‐wk rats. TGF‐β type II receptor (TβRII) protein demonstrated only cytoplasmic reactivity, while p38 MAPK protein and c‐jun protein showed both nuclear and cytoplasmic reactivity. The p38 MAPK nuclear reaction did not differ significantly between the study groups of rats. In contrast, the cytoplasmic response of the p38 MAPK was dependent on the administration of both HFD in two time intervals to the groups of rats. Significant differences in the nuclear reaction for c‐jun were observed between the exanimated groups of rats. The c‐jun nuclear expression was significantly lower in the HFD 20‐wk rats than in the HFD 16‐wk rats.ConclusionA high fat diet caused an increase in LV fibrosis in SPRD rats. Additionally, extension by the next 4 weeks the duration of HFD intake did not increase the LV fibrosis area. Moreover, high fat diet in both intervals significantly influences the expression of p38 MAPK and JNK in the LV. However, demonstrating their potential involvement in the processes of interstitial myocardial fibrosis and left ventricular remodeling requires further research.Support or Funding InformationThis study was financially supported by statutory funds from the Medical University of Warsaw (1MA/N/2018).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Articular chondrocytes isolated from the knee and ankle joints of human tissue donors demonstrate similar redox-regulated MAP kinase and Akt signaling

To compare key intracellular redox-regulated signaling pathways in chondrocytes derived from knee joint femoral cartilage and ankle joint talar cartilage in order to determine if differences exist that might contribute to the lower prevalence of ankle osteoarthritis (OA). Femoral and talar chondrocytes were treated with H2O2 generators (menadione or 2-3-dimethoxy-1,4-napthoquinone (DMNQ), fragments of fibronectin (FN-f)) to stimulate MAP kinase signaling (MAPK), or with IGF-1 to stimulate the Akt signaling pathway. Hyperoxidation of the peroxiredoxins, used as a measure of redox status, and phosphorylation of proteins pertinent to MAPK (p38, ERK, JNK, c-Jun) and Akt (Akt, PRAS40) signaling cascades were detected by immunoblotting. Treatment of femoral and talar chondrocytes with menadione, DMNQ or FN-f led to a time dependent increase in extracellular-regulated kinase (ERK) and p38 phosphorylation. DMNQ and FN-f stimulation enhanced phosphorylation of JNK and its downstream substrate, c-Jun. Menadione treatment did not stimulate JNK activity but hyperoxidized the peroxiredoxins and inhibited IGF-1-induced Akt activation. In all experiments, chondrocytes derived from the femur and talar joints displayed comparable MAP kinase responses after treatment with various catabolic stimuli, as well as similar Akt signaling responses after IGF-1 treatment. MAP kinase and Akt signaling in response to factors that modulate the intracellular redox status were similar in chondrocytes from knee and ankle joints suggesting that redox signaling differences do not explain differences in OA prevalence. Talar chondrocytes, when isolated from their native matrix, can be used to examine redox-regulated cell signaling events relevant to OA in either joint.

Arterial Thrombosis Is Accompanied by Elevated Mitogen-Activated Protein Kinase (MAPK) and Cyclooxygenase-2 (COX-2) Expression via Toll-Like Receptor 4 (TLR-4) Activation by S100A8/A9.

BackgroundThe aim of this study was to determine the involvement of S100A8/A9 in the development of arterial thrombosis.Material/MethodsA total of 303 patients were enrolled in this study, with 110 having acute coronary syndrome (ACS) and 110 having coronary heart disease (CHD), and 83 subjects served as healthy blood donors. The concentrations of Toll-like receptor 4 (TLR-4), cyclooxygenase-2 (COX-2), and S100A8/A9 protein were determined in the sera of the participants and in peripheral blood mononuclear cells (PBMCs) derived from a rat carotid artery thrombosis model and in human aortic endothelial cells (HAECs). The mitogen-activated protein kinase (MAPK) inhibitor SB203580 and the TLR-4 blocker CLI-095 were used to investigate the role of the TLR-4-MAPK-COX2 signaling axis in thrombosis.ResultsThe levels of COX-2, TLR-4, and S100A8/A9 in the sera of patients with ACS and CHD were significantly higher than in healthy controls (P<0.05). S100A8/A9 expression was significantly correlated with TLR-4 and COX-2 in the ACS group and with TLR-4 in the CHD group. In the rat carotid thrombosis model, the expressions of TLR-4, COX-2, and p-p38 MAPK significantly increased until 14 days after thrombosis induction, whereas S100A8/A9 expression increased until day 7, but then decreased. Administration of SB203580 to rats reduced COX-2 expression in PBMCs after thrombosis induction, and incubation of HAECs with CLI-095 reduced their p-p38 MAPK and COX-2 response to S100A8/A9 stimulation.ConclusionsS100A8/A9 is upregulated after blood vessel injury and is enhanced in combination with TLR-4 COX-2 induction via p38 MAPK activation.

Identification of a biologically active fragment of ALK and LTK-Ligand 2 (augmentor-α)

Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,β) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor-receptor interactions.

Dysregulated Inflammation And MAPK Signaling In Aged Human Muscle Following Exercise-induced Damage

The remarkable capacity of skeletal muscle to adapt and repair following injury is attenuated with age. Studies in young organisms suggest that acute changes in both extrinsic and intrinsic factors in the muscle environment are critical in regulating reparative potential. PURPOSE: To uncover potential factors involved in the impaired regenerative response of aged human skeletal muscle, we comprehensively assessed the molecular stress response following muscle damage in young and old individuals. METHODS: 11 young (22.7 ± 2.25 yrs) and 8 physically active old (70.9 ± 7.5 yrs) subjects completed a bout of 300 lengthening contractions (LC) on a Biodex dynamometer. Functional tests were performed as an indirect assessment of muscle damage and muscle biopsies were taken pre-exercise and at 3, 24, and 72 hours post-exercise. High throughput multi- plexing bead assays were used to analyze biopsy samples for content of inflammatory cytokines and protein concentrations of the mitogen activated protein kinase (MAPK) signaling pathway. RESULTS: After the bout of LC’s, muscle damage was evident by the loss of isometric force production in both groups (Young: 54.11 ± 22 and Old: 33.86 ± 17%). Old muscle displayed higher expression of MCP-1 (group, p=0.019) that appeared at the later 24 and 72 hour time points. There was also a rapid increase in NF-kb activity in the old following the bout of lengthening contractions (group x time, p=0.05). In the old, p38 protein content increased significantly at the 3 hour time point (Young: 0.8 ± 0.09 vs Old: 1.70 ± 0.47 fold) before returning to pre-exercise levels (group x time, p=0.0043). Additionally, by 3 hours post-exercise total JNK protein levels increased only in the old (Young: 1.6 ± 0.26 vs Old: 5.1 ± 0.77 fold) and remained significantly elevated (Young: 0.76 ± 0.20 vs Old: 4.5 ± 0.46 fold) 24 hours post-exercise (group x time, p=0.038) before returning to pre-exercise levels. CONCLUSION: skeletal muscle of physically active older individuals is characterized by a dysregulated and asynchronous inflammatory and MAPK response, each of which may individually or collectively contribute to the deterioration of muscle repair mechanisms that accompanies aging.

4-Methylthio-3-butenyl isothiocyanate mediates nuclear factor (erythroid-derived 2)-like 2 activation by regulating reactive oxygen species production in human esophageal epithelial cells

4-Methylthio-3-butenyl isothiocyanate (MTBITC) extracted from daikon (Raphanus sativus), which shows antimutagenicity, may have applications as an effective chemopreventive agent in several cancers; however, few reports have described the associated mechanisms. We investigated whether MTBITC induced cytoprotective genes, including phase II enzymes, in Het-1A human esophageal epithelial cells. HMOX1, NQO1, and GCLC mRNA levels and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein levels were increased in Het-1A cells treated with 10 μM MTBITC. Reactive oxygen species (ROS) tended to increase when Het-1A cells were treated with MTBITC, and the increases in ROS and Nrf2 expression in the cells treated with MTBITC were completely abolished by treatment with N-acetyl-l-cysteine. We also examined the relationships between Nrf2 activation and mitogen-activated protein kinase (MAPK) signaling by western blot analysis. MTBITC induced extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 phosphorylation in Het-1A cells; however, MTBITC did not affect the relationship between Nrf2 activation and MAPK responses. In the present study, we found that MTBITC induced Nrf2 activation and cytoprotective genes via ROS production in Het-1A cells. These results suggest that MTBITC may have the potential for preventing esophageal carcinogenesis through modification of carcinogen metabolism by phase II enzyme induction via ROS production.

Shenfuqiangxin Capsule inhibits apoptosis through mitogen-activated protein kinase signal pathway in rats with cardio-renal syndrome induced by infrarenal aortic-clamping

OBJECTIVETo investigate the effect of Shenfuqiangxin capsule and the underlying mechanism on cardio-renal syndrome (CRS) in rats induced by infrarenal aortic-clamping after renal ischaemia. METHODSMale Wistar rats underwent infrarenal aortic-clamping after renal ischaemia or sham operation. The surviving CRS rats were divided randomly into three groups: CRS group (CRS + 10 mL · kg−1 · d−1 pure water by gavage), SFQX group (CRS +13.2 g crude drug · kg−1 · d−1 Shenfuqiangxin by gavage), and handle region peptide (HRP) group (CRS + 10mg/kg HRP by vein). Sham operation rats were given 10 mL · kg−1 · d−1 pure water. Treatments were given 8 weeks after surgery, which lasted for 4 weeks. The rats were detected for heart structure and function by transthoracic echocardiography. PPR mRNA was detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR). To determine whether the mitogen-activated protein kinase (MAPK) signal pathway is included in the heart and kidney protective function of Shenfuqiangxin capsule, MAPK related proteins such as posophorylated C-Jun amino terminal kinase (p-JNK), posophorylated extracellular signal-regulated kinase ½ (p-ERK1/2), posophorylated p38 (p-p38) were examined by Western Blot. Apoptosis in heart and kidney tissues were detected by dUTP Nick End Labeling staining. RESULTSShenfuqiangxin capsule alleviated myocardial apoptosis and inhibited PRR mRNA expression and p-JNK, p-ERK1/2, p-p38 proteins expression in CRS rats. CONCLUSIONAll the results suggest that Shenfuqiangxin capsule improves the injured heart and kidney function maybe through inhibition of MAPK response pathway.

Gene and miRNA expression signature of Lewis lung carcinoma LLC1 cells in extracellular matrix enriched microenvironment.

BackgroundThe extracellular matrix (ECM), one of the key components of tumor microenvironment, has a tremendous impact on cancer development and highly influences tumor cell features. ECM affects vital cellular functions such as cell differentiation, migration, survival and proliferation. Gene and protein expression levels are regulated in cell-ECM interaction dependent manner as well. The rate of unsuccessful clinical trials, based on cell culture research models lacking the ECM microenvironment, indicates the need for alternative models and determines the shift to three-dimensional (3D) laminin rich ECM models, better simulating tissue organization. Recognized advantages of 3D models suggest the development of new anticancer treatment strategies. This is among the most promising directions of 3D cell cultures application. However, detailed analysis at the molecular level of 2D/3D cell cultures and tumors in vivo is still needed to elucidate cellular pathways most promising for the development of targeted therapies. In order to elucidate which biological pathways are altered during microenvironmental shift we have analyzed whole genome mRNA and miRNA expression differences in LLC1 cells cultured in 2D or 3D culture conditions.MethodsIn our study we used DNA microarrays for whole genome analysis of mRNA and miRNA expression differences in LLC1 cells cultivated in 2D or 3D culture conditions. Next, we indicated the most common enriched functional categories using KEGG pathway enrichment analysis. Finally, we validated the microarray data by quantitative PCR in LLC1 cells cultured under 2D or 3D conditions or LLC1 tumors implanted in experimental animals.ResultsMicroarray gene expression analysis revealed that 1884 genes and 77 miRNAs were significantly altered in LLC1 cells after 48 h cell growth under 2D and ECM based 3D cell growth conditions. Pathway enrichment results indicated metabolic pathway, MAP kinase, cell adhesion and immune response as the most significantly altered functional categories in LLC1 cells due to the microenvironmental shift from 2D to 3D. Comparison of the expression levels of selected genes and miRNA between LLC1 cells grown in 3D cell culture and LLC1 tumors implanted in the mouse model indicated correspondence between both model systems.ConclusionsGlobal gene and miRNA expression analysis in LLC1 cells under ECM microenvironment indicated altered immune response, adhesion and MAP kinase pathways. All these processes are related to tumor development, progression and treatment response, suggesting the most promising directions for the development of targeted therapies using the 3D cell culture models.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2825-9) contains supplementary material, which is available to authorized users.

Coordinated regulation of arbuscular mycorrhizal fungi and soybean MAPK pathway genes improved mycorrhizal soybean drought tolerance.

Mitogen-activated protein kinase (MAPK) cascades play important roles in the stress response in both plants and microorganisms. The mycorrhizal symbiosis established between arbuscular mycorrhizal fungi (AMF) and plants can enhance plant drought tolerance, which might be closely related to the fungal MAPK response and the molecular dialogue between fungal and soybean MAPK cascades. To verify the above hypothesis, germinal Glomus intraradices (syn. Rhizophagus irregularis) spores and potted experiments were conducted. The results showed that AMF GiMAPKs with high homology with MAPKs from Saccharomyces cerevisiae had different gene expression patterns under different conditions (nitrogen starvation, abscisic acid treatment, and drought). Drought stress upregulated the levels of fungi and soybean MAPK transcripts in mycorrhizal soybean roots, indicating the possibility of a molecular dialogue between the two symbiotic sides of symbiosis and suggesting that they might cooperate to regulate the mycorrhizal soybean drought-stress response. Meanwhile, the changes in hydrogen peroxide, soluble sugar, and proline levels in mycorrhizal soybean as well as in the accelerated exchange of carbon and nitrogen in the symbionts were contributable to drought adaptation of the host plants. Thus, it can be preliminarily inferred that the interactions of MAPK signals on both sides, symbiotic fungus and plant, might regulate the response of symbiosis and, thus, improve the resistance of mycorrhizal soybean to drought stress.

17β-estradiol attenuates homocysteine-induced oxidative stress and inflammatory response as well as MAPKs cascade via activating PI3-K/Akt signal transduction pathway in Raw 264.7 cells.

Oxidative stress, inflammatory response, and mitogen-activated protein kinases (MAPKs) cascade are significant pathogenic factors of osteoporosis. It has been reported that elevated homocysteine (Hcy) may activate oxidative stress and reduce bone mineral density in post-menopausal osteoporosis. Moreover, hormone replacement therapy has been widely used in clinic to prevent and treat post-menopausal women with osteoporosis and osteoporotic fracture, but the molecular mechanisms and relevant signal transduction pathways underlying the action of Hcy remain unclear. In this study, we investigated the effects of 17β-estradiol (17β-E2) on the Hcy-induced oxidative stress, inflammatory response and MAPKs cascade, as well as the underlying signal transduction pathway in murine Raw 264.7 cells. The reactive oxygen species (ROS) was assessed by fluorospectrophotometry. The proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β were analyzed by double-immunofluorescence labeling and reverse transcriptase polymerase chain reaction assay, respectively. Furthermore, phosphorylation levels of MAPKs cascade were measured by western blot analysis. A specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, Wortmannin (1 μM) was employed to determine whether PI3-K/Akt signaling pathway mediated the 17β-E2's effect on Raw 264.7 cells. 17β-E2 markedly decreased the ROS production induced by Hcy, the expression of TNF-α and IL-1β at protein and mRNA levels, and down-regulated the phosphorylation of MAPKs (ERK1/2, JNK and p38). These suppressing effects of 17β-E2 on Hcy-induced changes were reversed by pretreatment with PI3-K inhibitor Wortmannin. The results indicate that 17β-estradiol may attenuate Hcy-induced oxidative stress, inflammatory response and up-regulation of MAPKs in Raw 264.7 cells via PI3-K/Akt signal transduction pathway.

Differential NF-&amp;kappa;B and MAPK activation underlies fluoride- and TPA-mediated CXCL8 (IL-8) induction in lung epithelial cells

Different toxic agents have a varying potential to induce the production of the proinflammatory chemokine, CXCL8 (interleukin [IL]-8), in lung cells. A critical question is which mechanisms determine the magnitude and persistence of the CXCL8 responses to different stimuli. To approach this, we compared the potential of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and sodium fluoride (NaF) to induce CXCL8 responses in A549 cells, with emphasis on the importance of nuclear factor kappa B (NF-κB)- and mitogen-activated protein kinase (MAPK) signaling. Notably, TPA induced a greater release of CXCL8 than did NaF. Furthermore, TPA induced a strong, rapid, but transient upregulation of CXCL8 messenger (m)RNA, whereas NaF induced a weaker, more delayed, but persistent upregulation. With respect to signaling, TPA led to an early, strong, and relatively transient extracellular signal-regulated kinase (ERK)1/2 phosphorylation, and a less marked and even more transient phosphorylation of c-jun-N-terminal kinases (JNK1/2) and p38. In contrast, NaF elicited a lower, but relatively sustained increase in phosphorylation of ERK1/2, and a marked phosphorylation of p38 and JNK1/2, with the JNK1/2 response as most transient. Only ERK1/2 inhibition affected the TPA response, whereas inhibition of all the three MAPK cascades reduced NaF-induced CXCL8 release. TPA also induced an early, marked phosphorylation/translocation of p65 (NF-κB), whereas NaF induced slower, less pronounced effects on p65. The CXCL8 responses by TPA and NaF were reduced by p65-siRNA. In conclusion, all MAPK cascades were involved in NaF-induced CXCL8 release, whereas only ERK1/2 activation was involved in response to TPA. Furthermore, NF-κB activation appeared to be indispensable for CXCL8 induction. The early response, magnitude, and persistency of MAPK and NF-κB signaling seemed to be critical determinants for the potential to induce CXCL8. These findings underscore that a strong, rapid, and relatively transient activation of ERK1/2 in combination with NF-kB may be sufficient for a strong induction of CXCL8, which may exceed the effects of a more moderate ERK1/2 activation in combination with activation of p38, JNK1/2, and NF-κB.