Activation Of Mitogen-activated Protein Research Articles

Dynamic distress calls: volatile info chemicals induce and regulate defense responses during herbivory.

Plants are continuously threatened by a plethora of biotic stresses caused by microbes, pathogens, and pests, which often act as the major constraint in crop productivity. To overcome such attacks, plants have evolved with an array of constitutive and induced defense mechanisms- morphological, biochemical, and molecular. Volatile organic compounds (VOCs) are a class of specialized metabolites that are naturally emitted by plants and play an important role in plant communication and signaling. During herbivory and mechanical damage, plants also emit an exclusive blend of volatiles often referred to as herbivore-induced plant volatiles (HIPVs). The composition of this unique aroma bouquet is dependent upon the plant species, developmental stage, environment, and herbivore species. HIPVs emitted from infested and non-infested plant parts can prime plant defense responses by various mechanisms such as redox, systemic and jasmonate signaling, activation of mitogen-activated protein (MAP) kinases, and transcription factors; mediate histone modifications; and can also modulate the interactions with natural enemies via direct and indirect mechanisms. These specific volatile cues mediate allelopathic interactions leading to altered transcription of defense-related genes, viz., proteinase inhibitors, amylase inhibitors in neighboring plants, and enhanced levels of defense-related secondary metabolites like terpenoids and phenolic compounds. These factors act as deterrents to feeding insects, attract parasitoids, and provoke behavioral changes in plants and their neighboring species. This review presents an overview of the plasticity identified in HIPVs and their role as regulators of plant defense in Solanaceous plants. The selective emission of green leaf volatiles (GLVs) including hexanal and its derivatives, terpenes, methyl salicylate, and methyl jasmonate (MeJa) inducing direct and indirect defense responses during an attack from phloem-sucking and leaf-chewing pests is discussed. Furthermore, we also focus on the recent developments in the field of metabolic engineering focused on modulation of the volatile bouquet to improve plant defenses.

Isoflavones daidzin and daidzein inhibit lipopolysaccharide-induced inflammation in RAW264.7 macrophages

BackgroundInflammation contributes to various diseases and soybeans and legumes are shown to reduce inflammation. However, the bioactive ingredients involved and mechanisms are not completely known. We hypothesized that soy isoflavones daidzin and daidzein exhibit anti-inflammatory effect in lipopolysaccharides (LPS)-stimulated RAW264.7 macrophage cell model and that activation mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways may mediate the effect.MethodsCell viability and nitric oxide (NO) level were determined by 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Griess reagent respectively. ELISA kits and Western blotting respectively assessed the generations of pro-inflammatory cytokines and protein expressions of signaling molecules. p65 nuclear translocation was determined by immunofluorescence assay.ResultsThe in vitro results showed that both isoflavones did not affect cell viability at the concentrations being tested and significantly reduced levels of NO, pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), and inflammatory indicators such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in RAW264.7 cells. Daidzin and daidzein partially suppressed MAPK signaling pathways, reducing the phosphorylation of p38 and ERK; whilst phosphorylation of JNK was mildly but not significantly decreased. For the involvement of NF-κB signaling pathways, daidzin only reduced the phosphorylation of p65 whereas daidzein effectively inhibited the phosphorylation of IKKα/β, IκBα and p65. Daidzin and daidzein inhibited p65 nuclear translocation, comparable with dexamethasone (positive control).ConclusionThis study supports the anti-inflammatory effects of isoflavones daidzin and daidzein, which were at least partially mediated through inactivation of MAPK and/or NF-κB signaling pathways in macrophages.

Nitroglycerin-induced downregulation of AKT- and ERK1/2-mediated radiation-sensitive 52 expression to enhance pemetrexed-induced cytotoxicity in human lung cancer cells.

Nitroglycerin (NTG)-a nitric oxide-donating drug-is traditionally administered via the sublingual route to treat acute myocardial angina attacks. NTG also increases tumor blood flow and, consequently, cancer drug delivery to tumor cells. In the homologous recombination pathway, radiation-sensitive 52 (Rad52) plays a crucial role in DNA repair by promoting the annealing of complementary single-stranded DNA and stimulating radiation-sensitive 51 (Rad51) recombinase activity. Pemetrexed-a multitargeted antifolate agent-exhibits satisfactory clinical activity in wild-type nonsquamous non-small-cell lung cancer (NSCLC) cells. However, the synergistic activity of combination therapy with NTG and pemetrexed against NSCLC cells has not yet been clarified. In 2 NSCLC cell lines (i.e. lung squamous cell carcinoma H520 and lung adenocarcinoma H1975 cells), NTG reduced Rad52 expression; in addition, decreased phospho-AKT and phospho-ERK1/2 protein levels were observed. Enhancement of AKT or ERK1/2 activity through transfection with a constitutively active AKT (AKT-CA) vector or constitutively active mitogen-activated protein kinase kinase 1 (MKK1-CA) vector increased the Rad52 protein level and cell survival, which were suppressed by NTG. The knockdown of Rad52 expression by using small interfering RNA or by inhibiting AKT and ERK1/2 activity enhanced the cytotoxicity and cell growth inhibition induced by NTG. Moreover, NTG synergistically enhanced the cytotoxicity and cell growth inhibition induced by pemetrexed in NSCLC cells; these effects were associated with AKT and ERK1/2 inactivation and, consequently, Rad52 downregulation in H520 and H1975 cells. The results provide a rationale for combining NTG and pemetrexed in lung cancer treatment to improve lung cancer control.

Proliferative cells in racemose neurocysticercosis have an active MAPK signalling pathway and respond to metformin treatment

Racemose neurocysticercosis is an aggressive infection caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord, resulting in the displacement of the surrounding host tissue and chronic inflammation. We previously demonstrated that the continued growth of the racemose bladder wall is associated with the presence of mitotically active cells but the nature and control of these proliferative cells are not well understood. Here, we demonstrated by immunofluorescence that the racemose cyst has an active mitogen-activated protein kinases (MAPK) signalling pathway that is inhibited after treatment with metformin, which reduces racemose cell proliferation in vitro, and reduces parasite growth in the murine model of Taenia crassiceps cysticercosis. Our findings indicate the importance of insulin receptor-mediated activation of the MAPK signalling pathway in the proliferation and growth of the bladder wall of the racemose cyst and its susceptibility to metformin action. The antiproliferative action of metformin may provide a new therapeutic approach against racemose neurocysticercosis.

Ddx20, DEAD box helicase 20, is essential for the differentiation of oligodendrocyte and maintenance of myelin gene expression.

Oligodendrocytes form myelin sheaths that surround axons, contributing to saltatory conduction and proper central nervous system (CNS) function. Oligodendrocyte progenitor cells (OPCs) are generated during the embryonic stage and differentiate into myelinating oligodendrocytes postnatally. Ddx20 is a multifunctional, DEAD-box helicase involved in multiple cellular processes, including transcription, splicing, microRNA biogenesis, and translation. Although defects in each of these processes result in abnormal oligodendrocyte differentiation and myelination, the involvement of Ddx20 in oligodendrocyte terminal differentiation remains unknown. To address this question, we used Mbp-Cre mice to generate Ddx20 conditional knockout (cKO) mice to allow for the deletion of Ddx20 from mature oligodendrocytes. Mbp-Cre;Ddx20 cKO mice demonstrated small body sizes, behavioral abnormalities, muscle weakness, and short lifespans, with mortality by the age of 2months old. Histological analyses demonstrated significant reductions in the number of mature oligodendrocytes and drastic reductions in the expression levels of myelin-associated mRNAs, such as Mbp and Plp at postnatal day 42. The number of OPCs did not change. A thin myelin layer was observed for large-diameter axons in Ddx20 cKO mice, based on electron microscopic analysis. A bromodeoxyuridine (BrdU) labeling experiment demonstrated that terminal differentiation was perturbed from ages 2 weeks to 7 weeks in the CNS of Mbp-Cre;Ddx20 cKO mice. The activation of mitogen-activated protein (MAP) kinase, which promotes myelination, was downregulated in the Ddx20 cKO mice based on immunohistochemical detection. These results indicate that Ddx20 is an essential factor for terminal differentiation of oligodendrocytes and maintenance of myelin gene expression.

A putative exosporium lipoprotein GBAA0190 of Bacillus anthracis as a potential anthrax vaccine candidate

BackgroundBacillus ancthracis causes cutaneous, pulmonary, or gastrointestinal forms of anthrax. B. anthracis is a pathogenic bacterium that is potentially to be used in bioterrorism because it can be produced in the form of spores. Currently, protective antigen (PA)-based vaccines are being used for the prevention of anthrax, but it is necessary to develop more safe and effective vaccines due to their prolonged immunization schedules and adverse reactions.MethodsWe selected the lipoprotein GBAA0190, a potent inducer of host immune response, present in anthrax spores as a novel potential vaccine candidate. Then, we evaluated its immune-stimulating activity in the bone marrow-derived macrophages (BMDMs) using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Protective efficacy of GBAA0190 was evaluated in the guinea pig (GP) model.ResultsThe recombinant GBAA0190 (r0190) protein induced the expression of various inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in the BMDMs. These immune responses were mediated through toll-like receptor 1/2 via activation of mitogen-activated protein (MAP) kinase and Nuclear factor-κB (NF-κB) pathways. We demonstrated that not only immunization of r0190 alone, but also combined immunization with r0190 and recombinant PA showed significant protective efficacy against B. anthracis spore challenges in the GP model.ConclusionsOur results suggest that r0190 may be a potential target for anthrax vaccine.

Liver X Receptor Exerts Anti-Inflammatory Effects in Colonic Epithelial Cells via ABCA1 and Its Expression Is Decreased in Human and Experimental Inflammatory Bowel Disease.

Liver X receptor (LXR) exerts anti-inflammatory effects in macrophages. The aim of this study was to explore the expression and function of LXR in the colonic epithelium under inflammatory conditions. The expression of LXR was explored by Western blot and immunohistochemistry in colonic biopsies from patients diagnosed with inflammatory bowel disease (IBD) and control patients. In addition, LXR and its target gene expression were analyzed in the colon from interleukin (IL)-10-deficient (IL-10-/-) and wild-type mice. Caco-2 cells were pretreated with the synthetic LXR agonist GW3965 and further challenged with IL-1β, the expression of IL-8 and chemokine (C-C motif) ligand (CCL)-28 chemokines, the activation of mitogen-activated protein (MAP) kinases, and the nuclear translocation of the p65 subunit of nuclear factor kappa B was evaluated. Glibenclamide was used as an ABCA1 antagonist. We found that LXR expression was downregulated in colonic samples from patients with IBD and IL-10-/- mice. The nuclear positivity of LXR inversely correlated with ulcerative colitis histologic activity. Colonic IL-1β mRNA levels negatively correlated with both LXRα and LXRβ in the colon of IL-10-/- mice, where a decreased mRNA expression of the LXR target genes ABCA1 and FAS was shown. In addition, IL-1β decreased the expression of the LXR target gene ABCA1 in cultured intestinal epithelial cells. The synthetic LXR agonist GW3965 led to a decreased nuclear positivity of the p65 subunit of nuclear factor kappa B, a phosphorylation ratio of the p44-42 MAP kinase, and the expression of CCL-28 and IL-8 in IL-1β-stimulated Caco-2 cells. The pharmacological inhibition of ABCA1 increased the phosphorylation of p44-42 after GW3965 treatment and IL-1β stimulation. The LXR-ABCA1 pathway exerts anti-inflammatory effects in intestinal epithelial cells and is impaired in the colonic mucosa of patients with IBD and IL-10-/- mice.

Capsaicin Acts Through Reducing P38 MAPK-Dependent Thymidylate Synthase Expression to Enhance 5-Fluorouracil-Induced Cytotoxicity in Human Lung Cancer Cells

Capsaicin, an ingredient of green and red bell peppers, shows anticancer activity in several malignant cell lines. Thymidylate synthase (TS) is a well-validated anticancer drug target in non-small cell lung cancer (NSCLC) cells. However, whether capsaicin and 5-fluorouracil (5-FU) induce synergistic cytotoxicity in NSCLC cells by regulating TS expression is unclear. This study investigated the cytotoxicity of capsaicin and 5-FU co-treatment on two hoursuman lung adenocarcinoma cell lines, H520 and H1703, and the underlying mechanisms. Capsaicin decreased TS expression in a p38 mitogen-activated protein kinase (MAPK) inactivation–dependent manner in H520 and H1703 cells. Enhancement of p38 MAPK activity by transfection with constitutive active mitogen-activated protein kinase kinase six vectors increased TS expression and cell survival. In addition, capsaicin and 5-FU co-treatment enhanced synergistic cytotoxicity and inhibited cell growth associated with TS downregulation and p38 MAPK inactivation in H520 and H1703 cells. Capsaicin and 5-FU co-treatment did not affect the cellular content of capsaicin. These results show that capsaicin may be combined with 5-FU to treat NSCLC.

Gain-of-function hot spot mutant p53R248Q regulation of integrin/FAK/ERK signaling in esophageal squamous cell carcinoma

PurposeTP53, encoding the protein p53, is among the most frequently mutated genes in all cancers. A high frequency of 60 – 90% mutations is seen in esophageal squamous cell carcinoma (ESCC) patients. Certain p53 mutants show gain-of-function (GoF) oncogenic features unrelated to its wild type functions. MethodsThis study functionally characterized a panel of p53 mutants in individual ESCC cell lines and assayed for GoF oncogenic properties. ResultsThe ESCC cell line with endogenous p53R248Q expression showed suppressed tumor growth in an immunocompromised mouse model and suppressed colony growth in in vitro three-dimensional culture, when depleted of the endogenous p53 protein expression. This suppression is accompanied by suppressed cell cycle progression, along with reduced integrin expression and decreased focal adhesion kinase and extracellular-regulated protein kinase signaling and can be compensated by expression of a constitutively active mitogen-activated protein. P53R248Q enhances cell proliferation upon glutamine deprivation, as compared to other non-GoF mutants. ConclusionsIn summary, study of the functional contributions of endogenous p53 mutants identified a novel GoF mechanism through which a specific p53 mutant exerts oncogenic features and contributes to ESCC tumorigenesis.

Enhancement of Cartilage Regeneration of Synovial Stem Cells/Hydrogel by Using Transglutaminase-4.

Although mesenchymal stem cells (MSCs) transplantation is reportedly a promising strategy for repairing damaged articular cartilage, MSCs-based cartilage tissue engineering has numerous limitations, including poor implanted cell adhesion, phenotypic alteration of cells, regulation of mechanical properties, and engraftment rates after implantation. This study aimed to investigate the efficacy of transplantation of synovium-derived mesenchymal stem cells (SDSCs) encapsulated in a hyaluronic acid/collagen/fibrinogen (HA/COL/FG) composite gel by supplementing recombinant human transglutaminase 4 (rhTG-4) in treating osteochondral defects. RhTG-4 treatment induced the expression of integrin β1 and dynamic actin fiber, enhancing SDSCs adhesion to fibronectin. Supplementation of rhTG-4 significantly induced the proliferation of SDSCs encapsulated in the HA/COL/FG composite gel and increased the hardness of the extracellular matrix. Furthermore, supplementation of rhTG-4 significantly upregulated aggrecan and type II collagen mRNA. Pretreatment with integrin β1 siRNA markedly suppressed TG4-induced actin remodeling, activation mitogen-activated protein kinase (MAPK), and eventually the chondrogenesis-related genes. Moreover, transplantation of SDSCs encapsulated in HA/COL/FG/rhTG-4 composite gel in vivo yielded reconstructed tissue resembling native hyaline cartilage. These data suggest that rhTG-4 enhances cartilage regeneration of the SDSCs encapsulated in hydrogel in rabbits. Impact statement In this study, we investigated the effects of recombinant human transglutaminase 4 on the ability of synovium-derived mesenchymal stem cells encapsulated in a hyaluronic acid/collagen/fibrinogen composite gel to repair osteochondral defects. We believe that our study makes a significant contribution to the literature because it explores a method of improving an existing modality to mediate tissue repair.

Ets1 functions partially redundantly with Etv2 to promote embryonic vasculogenesis and angiogenesis in zebrafish

ETS transcription factors play an important role in the specification and differentiation of endothelial cells during vascular development. Despite previous studies, the role of the founding member of the ETS family, Ets1, in vascular development in vivo is only partially understood. Here, we generated a zebrafish ets1 mutant by TALEN genome editing and tested functional redundancy between Ets1 and a related ETS factor Etv2/Etsrp/ER71. While zebrafish ets1−/− mutants have a normal functional vascular system, etv2−/−;ets1-/embryos had more severe angiogenic defects and lower expression levels of kdr and kdrl, the two zebrafish homologs of the mammalian Vascular Endothelial Growth Factor Receptor 2 VEGFR2/Flk1, than etv2−/−embryos. Expression of constitutively active Mitogen-Activated Protein Kinase1 (MAP2K1) within endothelial cells partially rescued this angiogenic defect. Interestingly, ets1−/− embryos displayed extensive apoptosis within the trunk vasculature despite exhibiting normal vascular patterning. Loss of Ets1 combined with a partial knockdown of Etv2 function resulted in a decrease in endothelial cell numbers in the axial vasculature, which argues for a role of Ets1 in promoting vasculogenesis. We also demonstrate that although both Ets1 and Etv2 can induce ectopic vascular marker expression in zebrafish embryos, Ets1 activity is dependent on MAPK-mediated phosphorylation of its Thr30 and Ser33 residues, while Etv2 activity is not. Together, our results identify a novel function of Ets1 in regulating endothelial cell survival during vasculogenesis in vivo. Based on these findings, we propose a revised model of how Ets1 and Etv2 play unique and partially redundant roles to promote vascular development.

Pengaruh Penambahan PEG 4000 terhadap Stabilitas Fisik Sediaan Pembersih Wajah dengan Basis Salep Larut dalam Air mengandung Nanoemulsi M/A Minyak Biji Anggur (Vitis vinifera)

Abstrak: Radiasi sinar UV merupakan salah satu penyebab terjadinya photoaging. Minyak biji anggur merupakan salah satu bahan alam yang mengandung antioksidan dengan mekanisme penghambatan aktivasi Mitogen-Activated Protein Kinase (MAPK) dan jalur Nuclear Factor kB (NF-kB). Penelitian ini ditujukan untuk mengetahui pengaruh penambahan PEG 4000 terhadap stabilitas fisik sediaan pembersih wajah basis salep larut dalam air mengandung nanoemulsi m/a minyak biji anggur. Formulasi pembersih wajah basis salep larut dalam air dibuat dengan variasi konsentrasi PEG 4000 (13%, 17% dan 20%). Evaluasi sediaan pembersih wajah basis salep larut dalam air yang dilakukan meliputi uji organoleptik, pH, viskositas, daya sebar, dan uji stabilitas. Hasil evaluasi sediaan menunjukan bahwa F2 yang mengandung nanoemulsi m/a minyak biji anggur (Minyak Biji Anggur 1%, Tween 80 22,5 %, Gliserin 7,5% dan Aquademineral 69 %) sebanyak 33,5 %, PEG 4000 17 %, PEG 400 20 %, Asam stearat 13 % , TEA (Triethanolamin) 3 % dan Triethylhexanoin ad 100% merupakan formula terbaik dengan hasil kemampuan penyebaran 0,1828 ± 0,02 cm/g, pH 8,48 ± 0,01 dan viskositas 460.00 ± 20.00 dPas. Berdasarkan hasil evaluasi dapat dinyatakan bahwa F2 memiliki efektivitas pembersih wajah yang baik dan dari hasil uji stabilitas dapat dinyatakan bahwa F2 stabil secara fisik.
 Kata kunci: minyak biji anggur, nanoemulsi, pembersih wajah, basis salep larut dalam air

Involvement of A3 Adenosine Receptor in Neuroblastoma Progression via Modulation of the Hypoxic/Angiogenic Pathway.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The clinical course may range from spontaneous regression towards ganglioneuroblastoma/ganglioneuroma or maturation to a very aggressive form characterized by an extensive hypoxic area. In solid tumors, extracellular microenvironment hypoxia induces the transcription of hypoxia-inducible factors (HIFs) leading to synthesis of pro-angiogenic factor, VEGF; also, it increases extracellular adenosine production from ATP breakdown. To date, the role of this nucleoside in the hypoxic/angiogenic pathway characterizing the core of cancer mass has not been investigated yet. Therefore, the aim of the present study was to analyze the adenosine effect on modulation of the HIF-1α/2α/VEGF pathway mediated through A3 AR binding. To this end, we have used a selective A3 AR agonist IB-MECA or antagonist VUF 5574 in an in vitro model of malignant undifferentiated and all-trans retinoic acid (RA)-differentiated SH-SY5Y cells, representing the benign form of NB. Our results have shown that specific A3 AR stimulation induces HIF and VEGF expression through the activation of mitogen-activated protein kinase/Erk kinase signaling cascade. In conclusion, the data suggest that A3 AR may represent a marker of NB malignancy as well as a drug target for treatment of this solid tumor. Graphical Abstract.

RETRACTED ARTICLE: Sirt3 suppresses calcium oxalate-induced renal tubularepithelial cell injury via modification of FoxO3a-mediated autophagy

High oxalic acid and calcium oxalate (CaOx)-induced renal tubularepithelial cell (TEC) injury plays a key role in nephrolithiasis. However, themechanism remains unknown. Gene array analysis of the mice nephrolithiasis modelindicated significant downregulation of sirtuin 3 (Sirt3) and activation ofmitogen-activated protein kinase (MAPK) pathway. Kidney biopsy tissues of renalcalculi patients also showed decreased Sirt3 expression. Silencing Sirt3 exacerbatedoxidative stress and TEC death under CaOx stimulation. Restoring Sirt3 expression byoverexpression or enhancing its activity protected renal function and reduced TECdeath both in vitro and in vivo. Inhibiting the MAPK pathway resulted inupregulation of Sirt3 expression, preservation of renal function and decreased celldeath both in vitro and in vivo. Furthermore, Sirt3 could upregulate FoxO3a activitypost-translationally via deacetylation, dephosphorylation and deubiquitination.FoxO3a was found to interact with the promoter region of LC3B and to increase itsexpression, enhancing TEC autophagy and suppressing cell apoptosis and necrosis.Taken together, our results indicate that the MAPK/Sirt3/FoxO3a pathway modulatesrenal TEC death and autophagy in TEC injury.

MiRNA-575 suppresses angiogenesis by targeting Rab5-MEK-ERK pathway in endothelial cells.

Hypertension is a major risk factor for the development of atherosclerosis. Increased carotid intima-media thickness (CIMT) is generally considered as an early marker of atherosclerosis. Recently, circulating miRNAs have been implicated both as sensitive biomarkers and key regulators in the development of atherosclerosis. However, the biological functions and molecular regulatory mechanisms for miR-575 on angiogenesis remain unknown. In our study, we first identified up-regulation of circulating miR-575 in plasma of essential hypertensive patients with increased CIMT (iCIMT) compared with those patients with normal CIMT (nCIMT). Furthermore, the overexpression of miR-575 in human umbilical vein endothelial cells (HUVECs) by its mimics significantly inhibited migration and proliferation as well as induction of apoptosis of HUVECs. Inhibition of miR-575 performed the reverse effects of HUVECs. We further suggested Rab5B was the downstream target of miR-575 and knockdown of Rab5B significantly inhibited migration and proliferation of HUVECs. Overexpression of Rab5B largely rescued the miR-575-mediated impairment of angiogenesis processes including: cell proliferation, migration, and apoptosis as well as activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK-ERK) signaling. Therefore, our results uncover a novel role of miR-575 in endothelial cells, implying a potential biomarker and clinical target for atherosclerosis in hypertensive patients.

Maduramicin induces cardiac muscle cell death by the ROS-dependent PTEN/Akt-Erk1/2 signaling pathway.

Maduramicin (Mad), a polyether ionophore antibiotic, has been reported to be toxic to animals and humans because of being used at high doses or for long time, resulting in heart failure. However, the toxic mechanism of Mad in cardiac muscle cells is not well understood. Here, we show that Mad induced cell viability reduction and apoptosis in cardiac-derived H9c2, HL-1 cells, primary cardiomyocytes, and murine cardiac muscles, which was because of the inhibition of extracellular-signal-regulated kinase 1/2 (Erk1/2). Expression of constitutively active mitogen-activated protein kinase kinase 1 (MKK1) attenuated Mad-induced cell death in H9c2 cells, whereas silencing Erk1/2 or ectopic expression of dominant negative MKK1 strengthened Mad-induced cell death. Moreover, we found that both phosphatase and tensin homolog on chromosome 10 (PTEN) and protein kinase B (Akt) were implicated in the regulation of Erk1/2 inactivation and apoptosis in the cells and tissues exposed to Mad. Overexpression of dominant negative PTEN and/or constitutively active Akt, or constitutively active Akt and/or constitutively active MKK1 rescued the cells from Mad-induced dephosphorylated-Erk1/2 and cell death. Furthermore, Mad-induced reactive oxygen species (ROS) activated PTEN and inactivated Akt-Erk1/2 contributing to cell death, as N-acetyl- L-cysteine ameliorated the event. Taken together, the results disclose that Mad inhibits Erk1/2 via ROS-dependent activation of PTEN and inactivation of Akt, leading to cell death in cardiac muscle cells. Our findings suggest that manipulation of the ROS-PTEN-Akt-Erk1/2 pathway may be a potential approach to prevent Mad-induced cardiotoxicity.

Tu1273 - Expression of Constitutively Active Mitogen-Activated Protein Kinase 1 (MEK1) Profoundly Disrupts Ileal Epithelial Homeostasis

Tu1273 - Expression of Constitutively Active Mitogen-Activated Protein Kinase 1 (MEK1) Profoundly Disrupts Ileal Epithelial Homeostasis

Consumption of Broccoli Sprouts Attenuates Intracellular P38 Map Kinase and Reactive Oxygen Species Pro-Inflammatory Activation in Human Leukocytes: A Randomised- Controlled Trial

Dietary consumption of green vegetables has a protective association with reduction in cardiovascular diseases. The thiocyanate compound sulforaphane, which is obtained from green vegetables, induces antioxidant genes and can protect against pro-inflammatory cellular activation. Our objective was to investigate the influence of sulforaphane on leukocyte pro-inflammatory activation and assess the impact of a sulforaphane-rich diet on leukocyte activation in vivo. Healthy human volunteers were randomised to consume either broccoli sprout homogenates (BSH) rich in sulforaphane or alfalfa sprout homogenates (ASH) as controls in a double-blind crossover trial. Blood was sampled prior to and then at 1 h, 6 h and 24 h post-homogenate consumption and analysed by flow cytometry for intracellular markers of leukocyte activation, reactive oxygen species (ROS), p38 mitogen activated protein (MAP) kinase phosphorylation and p65 nuclear factor kappa B (NF-κB) phosphorylation. Plasma levels of sulforaphane were determined by LC-MS methods. Six participants were recruited into the study. Plasma concentrations of sulforaphane were 30 ng/ml at one hour following BSH and undetectable following ASH consumption. In the control ASH group, ROS activation and p38 MAP kinase phosphorylation were enhanced one hour following consumption. This increase was attenuated in the BSH group (ROS, p<0.02: p38, p<0.001 comparison between groups). We conclude that ingestion of sulforaphane-rich homogenate can protect against pro-inflammatory activation in circulating leukocytes by attenuation of constitutive ROS and p38 MAP kinase.

Sirtuin 2 aggravates postischemic liver injury by deacetylating mitogen-activated protein kinase phosphatase-1.

Sirtuin 2 (Sirt2) is known to negatively regulate anoxia‐reoxygenation injury in myoblasts. Because protein levels of Sirt2 are increased in ischemia‐reperfusion (I/R)‐injured liver tissues, we examined whether Sirt2 is protective or detrimental against hepatic I/R injury. We overexpressed Sirt2 in the liver of C57BL/6 mice using a Sirt2 adenovirus. Wild‐type and Sirt2 knockout mice were subjected to a partial (70%) hepatic ischemia for 45 minutes, followed by various periods of reperfusion. In another set of experiments, wild‐type mice were pretreated intraperitoneally with AGK2, a Sirt2 inhibitor. Isolated hepatocytes and Kupffer cells from wild‐type and Sirt2 knockout mice were subjected to hypoxia‐reoxygenation injury to determine the in vitro effects of Sirt2. Mice subjected to I/R injury showed typical patterns of hepatocellular damage. Prior injection with Sirt2 adenovirus aggravated liver injury, as demonstrated by increases in serum aminotransferases, prothrombin time, proinflammatory cytokines, hepatocellular necrosis and apoptosis, and neutrophil infiltration relative to control virus‐injected mice. Pretreatment with AGK2 resulted in significant improvements in serum aminotransferase levels and histopathologic findings. Similarly, experiments with Sirt2 knockout mice also revealed reduced hepatocellular injury. The molecular mechanism of Sirt2's involvement in this aggravation of hepatic I/R injury includes the deacetylation and inhibition of mitogen‐activated protein kinase phosphatase‐1 and consequent activation of mitogen‐activated protein kinases. Conclusion: Sirt2 is an aggravating factor during hepatic I/R injury. (Hepatology 2017;65:225‐236).

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

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