u-0126 and pyrazolanthrone

To investigate the role of the ROS/MAPK signaling axis in mediating the inhibitory effect of eriocitrin on proliferation and migration of hepatocellular carcinoma SMMC-7721 cells.. SMMC-7721 cells were treated with different concentrations of eriocitrin for 24 h, and the changes in cell viability were detected with CCK-8 assay. The migration and invasion abilities of the treated cells were evaluated using Transwell and scratch healing assays, the cell proliferation was assessed with colony-forming assay, and changes in nuclear morphology were observed with DAPI staining. Western blotting was performed to examine the changes in the expressions of E-cadherin, N-cadherin, MMP-2, MMP-9, PARP, Pro-caspase 3, pJNK, p-P38, and p-ERK. The effect of eriocitrin on PARP cleavage in SMMC-7721 cells pretreated with ERK, JNK and P38 inhibitors (U0126, SB203580 and SP600125, respectively) was detected using Western blotting. The effect of treatment with Nacetyl-cysteine (NAC, 30 μmol/L) and eriocitrin (100, 200, and 300 μg/mL), alone or in combination, on reactive oxygen species (ROS) levels in the cells was examined using a DCFH-DA fluorescent probe.. Eriocitrin can suppress the proliferation and migration and promote apoptosis of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPKs signaling pathway.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Poly(ADP-ribose) Polymerase Inhibitors; Reactive Oxygen Species

This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not-e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased β3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine.

Topics: Animals; Butadienes; Neuronal Outgrowth; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Rats; Temperature

Osteoarthritis (OA) is a chronic, age‑related osteoarthropathy that causes a considerable decline in quality of life, as well as economic losses due to its high incidence and poor prognosis. Mitogen‑activated protein kinases (MAPKs) regulate multiple cellular processes, including proliferation, differentiation and apoptosis, in certain diseases, such as cancer, diabetes and Alzheimer's disease. The present study aimed to investigate the regulatory role of the MAPK signaling pathway in early‑stage OA. A rabbit model of early‑stage OA was induced by treatment with the enzyme papain. U0126 [an extracellular signal‑regulated kinase (ERK) inhibitor], SP600125 [a Jun NH2‑terminal kinase (JNK) inhibitor] and SB203580 (a p38 inhibitor) were administered to the rabbits via intra‑articular injection. The severity of OA was assessed by histological examination using H&E, toluidine blue and safranin‑O/fast green staining, as well by analyzing the glycosaminoglycan (GAG) content and determining the OA Research Society International (OARSI) score. Western blotting was used to detect the protein expression levels of matrix metalloproteinase‑3 (MMP3), ERK, phosphorylated (p)‑ERK, p38, p‑p38, JNK, p‑JNK, Beclin1, UNC‑51‑like kinase 1 (ULK1) and microtubule‑associated protein 1 light chain 3 (LC3)II/I. U0126, SP600125 or SB203580 treatment significantly decreased the OARSI scores and significantly increased the GAG levels in the cartilaginous tissues of OA model rabbits. These results indicated that the MAPK inhibitors reduced the severity of OA‑induced injury at the early stage. Western blotting results demonstrated that MAPK inhibition significantly decreased the protein expression levels of MMP3 in OA cartilage. The protective effect of MAPK inhibitors in OA was mediated via the activation of autophagy, as demonstrated by the increased protein expression levels of LC3II/I, ULK1 and Beclin1. Overall, the data indicated that MAPK inhibitors may exert a protective effect against OA by restoring compromised autophagy. Furthermore, the present study suggested that MAPK inhibitors may represent a potential pharmacological strategy for treating OA in the future.

Topics: Animals; Anthracenes; Autophagy; Butadienes; Disease Models, Animal; Imidazoles; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Osteoarthritis; Protein Kinase Inhibitors; Pyridines; Rabbits; Severity of Illness Index

The epithelial‑mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is an important underlying mechanism of proliferative vitreoretinopathy (PVR). We previously found that L‑carnitine (β‑hydroxy‑γ‑N-trimethylammonium‑butyrate, LC) was significantly lower during the transforming growth factor‑β1 (TGF‑β1)‑induced EMT process in ARPE‑19 cells. The present study assessed the role of LC in the EMT of RPE cells. The migration of RPE cells was detected using a Transwell migration assay. Then, EMT‑related biomarkers were measured via western blotting, immunofluorescence and reverse transcription‑quantitative PCR. It was observed that LC attenuated the TGF‑β1‑induced downregulation of the epithelial markers E‑Cadherin and zonula occludens‑1, as well as the expression of mesenchymal markers fibronectin and α‑smooth muscle actin. Meanwhile, LC blocked Erk1/2 and JNK pathways in the EMT of RPE cells. Moreover, treatment with a peroxisome proliferator‑activated receptor γ (PPARγ) inhibitor prevented the effect of LC on EMT. Taken together, these data suggested that LC attenuated EMT induced by TGF‑β1 via inhibition of the Erk1/2 and JNK pathways and upregulation of PPARγ expression.

Topics: Anilides; Anthracenes; Butadienes; Carnitine; Cell Movement; Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; MAP Kinase Signaling System; Models, Biological; NF-kappa B; Nitriles; PPAR gamma; Retinal Pigment Epithelium; Smad Proteins; Transforming Growth Factor beta1

Microtubule-targeting agents (MTAs) are the most effective chemotherapeutics used in cancer therapy to date, but their clinical use is often hampered by the acquisition of resistance. Thereby, elucidation of the molecular signaling pathways activated by novel FDA-approved MTAs such as eribulin is important for future therapeutic applications. In contrast to several reports, we show here that regardless of the presence of caspase-3, clinically relevant concentrations of eribulin and the classical MTA paclitaxel predominantly induce caspase-independent cell death in MCF-7 breast carcinoma cells. On the molecular level, several key proteins involved in apoptosis such as p53, Plk1, caspase-2, and Bim as well as the two MAPKs ERK and JNK were activated by both compounds to a similar extent. However, none of them proved to be important for eribulin- and paclitaxel-induced cytotoxicity, as their siRNA-mediated knockdown or inactivation by small molecule inhibitors did not alter cell death rates. In contrast, knockdown of the anti-apoptotic Bcl-2 protein, which becomes heavily phosphorylated at Ser70 during MTA treatment, resulted surprisingly in a reduction of MTA-mediated cell death. This phenomenon can be most likely explained by our observation that the absence of Bcl-2 slowed down cell cycle progression resulting in fewer cells entering mitosis, thereby delaying the mitotic capability of these MTAs to induce cell death. Taken together, although eribulin and paclitaxel disturb the mitotic spindle differently, they exhibit no functional differences in downstream molecular cell death signaling in MCF-7 breast cancer cells.

Topics: Anthracenes; Butadienes; Caspase 3; Cell Cycle; Cell Death; Cell Line, Tumor; Furans; Humans; Ketones; Microtubules; Mitogen-Activated Protein Kinases; Nitriles; Paclitaxel; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

It has been reported that miR-294 is highly expressed in hepatocellular carcinoma (HCC) tissues and cells. However, the potential role of miR-294 in the pathogenesis of HCC remains unclear. This study aimed to explore the role of miR-294 in HCC and the potential mechanism involved in this process.. Reverse transcription polymerase chain reaction was performed to determine the expression of miR-294 in HCC tissues and cell lines. Following the overexpression or knockdown of miR-294, the proliferation, migration, and invasion abilities of cells were determined using Cell Counting Kit-8 (CCK-8), wound healing and transwell assays, respectively. The phosphorylation of JNK and ERK was determined through western blotting. Furthermore, HCC cells were treated with JNK inhibitor SP600125 or ERK inhibitor U0126 and transfected with miR-294 mimics or negative control. Subsequently, the phosphorylation of JNK and ERK was evaluated and the proliferation, migration and invasion abilities of HCC cells were also determined.. The expression of miR-294 was significantly increased in HCC tissues and cell lines. Following the overexpression of miR-294, proliferation, migration, and invasion were promoted in the SSMC-7721 cell line, and the phosphorylation of JNK and ERK was increased, while silencing of miR-294 led to the opposite result. Use of the JNK or ERK inhibitor to treat SSMC-7721 cells transfected with miR-294 mimics decreased the phosphorylation of JNK and ERK and inhibited the proliferation, migration and invasion abilities of cells.. miR-294 is important for the development of HCC in terms of the biological activities of cells, and may be a novel therapeutic target for HCC.

Topics: Aged; Anthracenes; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; MAP Kinase Kinase 4; MAP Kinase Signaling System; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Nitriles; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Up-Regulation

Topics: Aged; Anthracenes; Apoptosis; Bortezomib; Butadienes; Caspases; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Kinase 4; MAP Kinase Signaling System; Middle Aged; Nitriles; p21-Activated Kinases; Receptors, TNF-Related Apoptosis-Inducing Ligand; Stomach Neoplasms; TNF-Related Apoptosis-Inducing Ligand

Ephrin A1 has a role in a variety of biological events, including cell proliferation, differentiation, migration, and angiogenesis. Ephrin A1 expression is abundant in trophoblasts and endometrial cells during the implantation period; however, its intracellular activities have not yet been reported in bovine endometrial (BEND) epithelial cells. The aim of this study was to identify the functional role of ephrin A1 in BEND cells, which have served as a good model system for investigating the regulation of signal transduction following treatment with interferon-τ (IFNT) in vitro. Supplementation of ephrin A1 to BEND cells increased cell proliferation and increased levels of proliferating cell nuclear antigen and cyclin D1 protein in BEND cell nuclei. To investigate intracellular mechanisms regulated by ephrin A1, we performed Western blot analysis focused on mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling, which are significantly involved in the successful maintenance of pregnancy. Ephrin A1 dose-dependently increased phosphorylation of extracellular signal-regulated kinases (ERK)1/2, c-Jun N-terminal kinases (JNK), P38, protein kinase B (AKT), P70S6K, S6, and cyclin D1, and the activated proteins were suppressed by pharmacological inhibitors including wortmannin (a PI3K inhibitor), U0126 (an ERK1/2 inhibitor), and SP600125 (a JNK inhibitor). Among ephrin A1 receptors, abundant expression of EPHA2 and EPHA4 messenger RNA was detected in BEND cells by reverse transcription polymerase chain reaction analysis. Furthermore, tunicamycin-induced endoplasmic reticulum (ER) stress was inactivated by ephrin A1 treatment of BEND cells. Our findings suggest that ephrin A1 promotes the development of BEND cells and likely enhances uterine capacity and maintenance of pregnancy by activating MAPK and PI3K signaling cascades and by restoring ER stress.

Topics: Animals; Anthracenes; Butadienes; Cattle; Cell Cycle; Cell Line; Cell Proliferation; Cyclin D1; Endometrium; Endoplasmic Reticulum Stress; Ephrin-A1; Extracellular Signal-Regulated MAP Kinases; Female; Interferon Type I; JNK Mitogen-Activated Protein Kinases; Nitriles; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Pregnancy; Pregnancy Proteins; Proliferating Cell Nuclear Antigen; Receptor, EphA2; Receptor, EphA4; Wortmannin

Endothelial diaphragms are subcellular structures critical for mammalian survival with poorly understood biogenesis. Plasmalemma vesicle associated protein (PLVAP) is the only known diaphragm component and is necessary for diaphragm formation. Very little is known about PLVAP regulation. Phorbol esters (PMA) are known to induce de novo PLVAP expression and diaphragm formation. We show that this induction relies on the de novo production of soluble factors that will act in an autocrine manner to induce PLVAP transcription and protein expression. We identified vascular endothelial growth factor-A (VEGF-A) signalling through VEGFR2 as a necessary but not sufficient downstream event as VEGF-A inhibition with antibodies and siRNA or pharmacological inhibition of VEGFR2 only partially inhibit PLVAP upregulation. In terms of downstream pathways, inhibition of MEK1/Erk1/2 MAP kinase blocked PLVAP upregulation, whereas inhibition of p38 and JNK MAP kinases or PI3K and Akt had no effect on PMA-induced PLVAP expression. In conclusion, we show that VEGF-A along with other secreted proteins act synergistically to up-regulate PLVAP in MEK1/Erk1/2 dependent manner, bringing us one step further into understanding the genesis of the essential structures that are endothelial diaphragms.

Topics: Anthracenes; Autocrine Communication; Axitinib; Butadienes; Flavonoids; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Imidazoles; Indazoles; MAP Kinase Kinase 1; MAP Kinase Kinase 4; Membrane Proteins; Nitriles; Phosphatidylinositol 3-Kinases; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; RNA, Small Interfering; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

STRIP2 (FAM40B) was reported to regulate tumor cell migration. Our study aims to discuss the effect of STRIP2 in mouse aortic smooth muscle cell (MOVAS) proliferation and migration processes, which contributes greatly to atherosclerosis formation. In MOVAS cells, STRIP2 depletion suppressed cell proliferation and migration, which were related to a remarkable decrease in matrix metalloproteinases-2 (MMP-2)/MMP-9 expression. Additionally, P38 mitogen-activated protein kinases and Protein kinase B (AKT) are inactivated while extracellular signal-regulated kinase (ERK1/2) and jun N-terminal kinase (JNK) are activated upon STRIP2 silencing. SB203580 (P38 inhibitor) further reduced AKT phosphorylation (p-AKT) while dehydrocorydaline chloride (Dc; P38 activator) reversed this effect. Furthermore, Dc significantly recovered MMP-2 expression in STRIP2-knockdown cells. As expected, overexpressing STRIP2 exhibited a contrary effect. Dc and AKT activator SC79 reversed the inhibition of cell proliferation and migration induced by STRIP2 silencing. Interestingly, STRIP2 depletion increased vascular endothelial growth factor level significantly. Taken together, STRIP2 contributed to cell proliferation and migration through P38-AKT-MMP-2 signaling in MOVAS cells, indicating the importance of STRIP2 in atherosclerosis.

Topics: Acetates; Alkaloids; Animals; Anthracenes; Axin Protein; Benzopyrans; Butadienes; Cell Line; Cell Movement; Cell Proliferation; Cytoskeletal Proteins; Gene Expression Regulation; Gene Knockdown Techniques; Gene Silencing; Imidazoles; Matrix Metalloproteinase 2; Mice; Mitomycin; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitriles; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Pyridines

Oxidative stress due to reactive oxygen species plays a central role in pathophysiology of neurodegenerative diseases. Inhibition of mitogen-activated protein kinase (MAPK) cascades attenuates the oxidative induced cell stress and behaves as potential neuroprotection agent.. In this study, we evaluate hydrogen peroxide induced neural cell stress and determine how different MAPK inhibitors restore the cell damage.. The results indicated that oxidative stress induced by neural cell damage commonly exists, and MAPK inhibitors partially and selectively attenuated the cell damage by reducing ROS production and cell apoptosis. The cultured neurons are more susceptible to hydrogen peroxide than subculture cells.. We conclude that the essential role of different MAPK inhibitors is to attenuate the hydrogen peroxide induced neuronal cell damage. Those data broaden the implication between individual neural cells and different MAPK inhibitors and give clues for oxidative stress induced neural diseases.

Topics: Animals; Anthracenes; Butadienes; Cell Survival; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Imidazoles; Mice; Mitogen-Activated Protein Kinase Kinases; Neurodegenerative Diseases; Neurons; Nitriles; Oxidative Stress; PC12 Cells; Primary Cell Culture; Pyridines; Rats; Reactive Oxygen Species

Multi-protein complexes, termed "inflammasomes," are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.

Topics: Adaptor Proteins, Signal Transducing; Animals; Anthracenes; Apoptosis Regulatory Proteins; Brain; Brain Ischemia; Butadienes; Extracellular Signal-Regulated MAP Kinases; Imidazoles; Inflammasomes; Mice; Neurons; NF-kappa B; Nitriles; NLR Family, Pyrin Domain-Containing 3 Protein; Pyridines; Signal Transduction; Stroke; Sulfones

We previously reported that 37-kDa laminin receptor precursor involved in metastasis of lung adenocarcinoma cancer cells. In this study, we further revealed that hypoxia induced 37-kDa laminin receptor precursor expression and activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in lung adenocarcinoma cancer cells. In addition, we further demonstrated that the c-Jun N-terminal kinase inhibitor SP600125 and extracellular signal-regulated protein kinase inhibitor U0126 blocked the c-Jun activity and abolished hypoxia-induced 37-kDa laminin receptor precursor expression and promoter activity in a concentration-dependent manner. However, the p38 mitogen-activated protein kinase inhibitor did not affect 37-kDa laminin receptor precursor expression and c-Jun activity in response to hypoxia. Furthermore, downregulated c-Jun expression by short interfering RNA could also inhibit hypoxia-induced 37-kDa laminin receptor precursor expression and transcriptional activity. The inhibition of 37-kDa laminin receptor precursor expression by SP600125 and U0126 could be rescued by c-Jun overexpression. Studies using luciferase promoter constructs revealed a significant increase in the activity of promoter binding in the cells exposed to hypoxia, which was lost in the cells with mutation of the activator protein 1 binding site. Electrophoresis mobility shift assay and chromatin immunoprecipitation demonstrated a functional activator protein 1 binding site within 37-kDa laminin receptor precursor gene regulatory sequence located at -271 relative to the transcriptional initiation point. Hypoxia-induced invasion of A549 cells was inhibited by the pharmacologic inhibitors of c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated protein kinase (U0126) as well as 37-kDa laminin receptor precursor-specific siRNA or antibody. Our results suggest that hypoxia-elicited c-Jun/activator protein 1 regulates 37-kDa laminin receptor precursor expression, which modulates migration and invasion of lung adenocarcinoma cells.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Anthracenes; Butadienes; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; MAP Kinase Kinase 4; MAP Kinase Signaling System; Nitriles; Phosphorylation; Receptors, Laminin

Macrophages are key players in the innate immune response. Turnover of phosphoinositides (PI), particularly phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2), has been implicated in macrophage functions such as toll-like receptor (TLR)-mediated cytokine production and phagocytosis. However, PI metabolizing enzymes responsible for macrophage functions are not well defined. The phospholipase C (PLC) family of enzymes is critical in PI(4,5)P2 turnover. In this study, we investigated the role of PLCδ1, a prototype PLC, in macrophages on the expression of inflammation-associated genes and phagocytosis. Lipopolysaccharides (LPS) signal through TLR4 to produce proinflammatory cytokines such as interleukin (IL)-1β. LPS stimulation of both RAW264.7 murine macrophages and murine bone marrow-derived macrophages resulted in lower PLCδ1 mRNA and protein expression levels, compared to that in the control. Using chemical inhibitor compounds, we demonstrated that the up-regulation of p38 MAPK activity led to down-regulation of PLCδ1 mRNA expression in macrophages. PLCδ1 reduction by RNAi or gene deletion resulted in greater LPS-induced IL-1β expression than that observed in the control siRNA-treated cells, without increasing TLR4 cell surface expression. PLCδ1 also negatively regulated LPS-induced cell spreading. Analysis of Fcγ receptor-mediated phagocytosis demonstrated an increased phagocytosis index after PLCδ1 knockdown in RAW264.7 cells. Conversely, overexpression of PLCδ1 reduced phagocytosis whereas catalytic inactive PLCδ1 had no effect. Altered levels of PLCδ1 affected the binding of opsonized latex beads with cells, rather than the phagocytic activity. Taken together, the data suggest that PLCδ1 negatively regulates LPS-induced production of IL-1β and Fcγ receptor-mediated phagocytosis in macrophages.

Topics: Animals; Anthracenes; Butadienes; Cell Line; Gene Expression Regulation; Imidazoles; Interleukin-1beta; Lipopolysaccharides; Macrophages; Mice; Mice, Knockout; Mutation; Nitriles; p38 Mitogen-Activated Protein Kinases; Phagocytosis; Phosphatidylinositol 4,5-Diphosphate; Phospholipase C delta; Primary Cell Culture; Protein Kinase Inhibitors; Pyridines; Receptors, IgG; RNA, Small Interfering; Signal Transduction; Toll-Like Receptor 4

Mutations in DJ-1, reactive gliosis and concomitant inflammatory processes are implicated in the pathogenesis and progression of Parkinson's disease (PD). To study the physiological consequences of DJ-1 mutation in the context of neuroinflammatory insult, primary cortical astrocytes were isolated from DJ-1 knockout mice. Astrocytes were exposed to 1μg/mL lipopolysaccharide (LPS) for 24h following 2h pre-exposure to inhibitors of MEK (U0126), JNK (JNK inhibitor II) or p38 (SB203580). Real-time PCR was used to assess the LPS-induced expression of pro-inflammatory mediators cyclooxygenase 2 (COX2), inducible nitric oxide synthetase (NOS2), and tumor necrosis factor α (TNFα). LPS-induced expression of COX2 decreased similarly in DJ-1(+/+) and DJ-1(-/-) astrocytes in response to inhibition of p38, but was unaffected by inhibition of MEK or JNK. No significant alterations in NOS2 expression were observed in any inhibitor-treated cells. The inhibitors did not affect expression of TNFα; however, DJ-1(-/-) astrocytes had consistently lower expression compared to DJ-1(+/+) counterparts. Secretion of TNFα and prostaglandin E2 (PGE2) into the culture medium was significantly decreased in DJ-1(-/-) astrocytes, and inhibition of p38 decreased this secretion in both genotypes. In conclusion, DJ-1(-/-) astrocytes may provide decreased neuroprotection to surrounding neurons due to alterations in pro-inflammatory mediator expression.

Topics: Animals; Anthracenes; Astrocytes; Butadienes; Cyclooxygenase 2; Dinoprostone; Imidazoles; Inflammation Mediators; Lipopolysaccharides; Mice; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type II; Nitriles; Primary Cell Culture; Protein Deglycase DJ-1; Pyridines; Tumor Necrosis Factor-alpha

Cadmium (Cd) is a highly toxic metal that affects the central nervous system. Recently we have demonstrated that inhibition of mTOR by rapamycin rescues neuronal cells from Cd-poisoning. Here we show that rapamycin inhibited Cd-induced mitochondrial ROS-dependent neuronal apoptosis. Intriguingly, rapamycin remarkably blocked phosphorylation of JNK, Erk1/2 and p38 in neuronal cells induced by Cd, which was strengthened by co-treatment with Mito-TEMPO. Inhibition of JNK and Erk1/2 by SP600125 and U0126, respectively, potentiated rapamycin's prevention from Cd-induced apoptosis. Consistently, over-expression of dominant negative c-Jun or MKK1 also potently improved the inhibitory effect of rapamycin on Cd neurotoxicity. Furthermore, pretreatment with SP600125 or U0126, or expression of dominant negative c-Jun or MKK1 enhanced the inhibitory effects of rapamycin or Mito-TEMPO on Cd-induced ROS. Further investigation found that co-treatment with Mito-TEMPO/rapamycin more effectively rescued cells by preventing Cd inactivation of PP2A than treatment with rapamycin or Mito-TEMPO alone. Over-expression of wild-type PP2A reinforced rapamycin or Mito-TEMPO suppression of activated JNK and Erk1/2 pathways, as well as ROS production and apoptosis in neuronal cells in response to Cd. The findings indicate that rapamycin ameliorates Cd-evoked neuronal apoptosis by preventing mitochondrial ROS inactivation of PP2A, thereby suppressing activation of JNK and Erk1/2 pathways. Our results underline that rapamycin may have a potential in preventing Cd-induced oxidative stress and neurodegenerative diseases.

Topics: Animals; Anthracenes; Apoptosis; Butadienes; Cadmium; Genes, jun; MAP Kinase Signaling System; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Neurons; Nitriles; PC12 Cells; Phosphorylation; Protein Phosphatase 2; Rats; Reactive Oxygen Species; Signal Transduction; Sirolimus

The migration of circulating mesenchymal stem cells (MSCs) to injured tissue is an important step in tissue regeneration and requires adhesion to the microvascular endothelium. The current study investigated the underlying mechanism of MSC adhesion to endothelial cells during inflammation. In in vitro MSC culture, tumor necrosis factor‑α (TNF‑α) increased the level of vascular cell adhesion molecule‑1 (VCAM‑1) expression in a dose‑dependent manner. The nuclear factor-κB (NF-κB), extracellular signal‑regulated kinase (ERK) and c‑Jun N‑terminal kinase (JNK) signaling pathway inhibitors, pyrrolidine dithiocarbamate (PDTC), U0126 and SP600125, respectively, suppressed VCAM‑1 expression induced by TNF‑α at the mRNA and protein levels (P<0.05). TNF‑α augmented the activation of NF‑κB, ERK and JNK, and promoted MSC adhesion to human umbilical vein endothelial cells; however, the inhibitors of NF‑κB, ERK and JNK did not affect this process in these cells. The results of the current study indicate that adhesion of circulating MSCs to the endothelium is regulated by TNF-α-induced VCAM-1 expression, which is potentially mediated by the NF‑κB, ERK and JNK signaling pathways.

Topics: Anthracenes; Biomarkers; Butadienes; Cell Adhesion; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Immunophenotyping; JNK Mitogen-Activated Protein Kinases; Mesenchymal Stem Cells; NF-kappa B; Nitriles; Phosphorylation; Signal Transduction; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

The leaves and stems of Perilla frutescens var. acuta Kudo (PF) have been used to prevent threatened abortion in traditional medicine in the East Asian countries. Because reduced receptivity of endometrium is a cause of abortion, we analyzed the action of PF on the endometrial receptivity. PF increased the level of leukemia inhibitory factor (LIF), a major cytokine regulating endometrial receptivity, and LIF receptor in human endometrial Ishikawa cells. The PF-induced LIF expression was mediated by c-jun N-terminal kinase (JNK) and p38 pathways. Adhesion between Ishikawa cells and trophoblastic JAr cells stimulated by PF treatment was abolished by knock down of LIF expression or antagonism of LIFR. In addition, the expressions of integrin β3 and β5 were increased by PF treatment in Ishikawa cells. The PF-induced expression of integrin β3 and β5 was reduced with an LIFR antagonist. Neutralization of both integrins successfully blocked PF-stimulated adhesion of JAr cells and Ishikawa cells. These results suggest that PF enhanced the adhesion between Ishikawa cells and JAr cells by increasing the expression of integrin β3 and β5 via an LIF-dependent pathway. Given the importance of endometrial receptivity in successful pregnancy, PF can be a novel and effective candidate for improving pregnancy rate.

Topics: Anthracenes; Butadienes; Cell Adhesion; Cells, Cultured; Endometrium; Female; Humans; Imidazoles; Integrin beta Chains; Leukemia Inhibitory Factor; Leukemia Inhibitory Factor Receptor alpha Subunit; MAP Kinase Signaling System; Nitriles; Perilla frutescens; Plant Extracts; Plant Leaves; Plant Roots; Pyridines; RNA, Small Interfering; Signal Transduction

Intraplaque angiogenesis has been recognized as an important risk factor for the rupture of advanced atherosclerotic plaques in recent years. CD147, also called Extracellular Matrix Metalloproteinase Inducer, has been found the ability to promote angiogenesis in many pathological conditions such as cancer diseases and rheumatoid arthritis via the up-regulation of vascular endothelial growth factor (VEGF), a critical mediator of angiogenesis. We investigated whether CD147 would also induce the up-regulation of VEGF in the foam cells formation process and explored the probable signaling pathway. The results showed the expression of CD147 and VEGF was significantly higher in U937-derived foam cells. After CD147 stealth siRNA transfection treatment, the production of VEGF was reduced depended on the inhibition efficiency of CD147 siRNAs.The special signaling pathway inhibitors LY294002, SP600125, SB203580 and U0126 were added to cultures respectively and the results showed LY294002 dose-dependently inhibited the expression of VEGF. The reduction of phospho-Akt was observed in both LY294002 and siRNA groups, suggested that the phosphatidylinositol 3-kinase/Akt pathway may be the probable signaling pathway underlying CD147 induced up-regulation of VEGF in U937-derived foam cells.

Topics: Anthracenes; Atherosclerosis; Basigin; Butadienes; Chromones; Flow Cytometry; Foam Cells; Gene Expression Regulation; Gene Silencing; Humans; Imidazoles; Lipoproteins, LDL; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Signal Transduction; U937 Cells; Up-Regulation; Vascular Endothelial Growth Factor A

To investigate the molecular mechanisms of nitric oxide (NO)-induced cytotoxic effect in human gingival fibroblast (HGF) cells.. After sodium nitroprusside (SNP), as NO donor, was treated to HGF, viability was measured by MTT assay and apoptosis was determined by TUNEL and DNA fragmentation assay. Mitochondrial membrane potential was detected using confocal microscopy, and caspase activity assay was measured by spectrophotometer. Mitogen-activated protein kinases (MAPK) activation, Bax/Bcl-2 ratio and cytochrome c release were analysed by Western blot analyses. Cells were exposed to MAPK inhibitors (U0126, SB203580 and SP600125) before SNP treatment to investigate the effects of MAPK kinases on the NO-induced apoptosis in HGF. Statistical analysis was performed using one-way analysis of variance with the Student-Newman-Keuls post hoc test for multiple group comparison.. Apoptosis was significantly increased (P = 0.011 and 0.0004, respectively) in the presence of SNP (1 and 3 mmol L(-1) ) after 12 h in HGF. However, 1H-[1,2,4] oxadiatolo [4, 3-a] cluinoxaline-1-one (ODQ), a soluble guanylate cyclase inhibitor, did not block the decrement of cell viability by NO. SNP treatment induced the loss of mitochondrial membrane potential, release of cytochrome c, increased Bax/Bcl-2 ratio and activation of caspases in HGF. Also, SNP treatment increased phosphorylation of MAPKinases and c-Jun N-terminal kinase (JNK) inhibitor (5 and 10 μmol L(-1) ) rescued cell viability decreased by SNP in HGF (P = 0.024 and 0.0149, respectively).. Nitric oxide induced apoptosis in human gingival fibroblast through the mitochondria-mediated pathway by regulation of Bcl-2 family and JNK activation.

Topics: Anthracenes; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Butadienes; Cell Survival; Cells, Cultured; Cytochromes c; Enzyme Inhibitors; Fibroblasts; Gingiva; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Membrane Potentials; Mitochondria; Nitric Oxide; Nitriles; Nitroprusside; Pyridines; Signal Transduction

Thioredoxin-interacting protein (TXNIP) is involved in various cellular processes including redox control, metabolism, differentiation, growth, and apoptosis. With respect to hematopoiesis, TXNIP has been shown to play roles in natural killer cells, dendritic cells, and hematopoietic stem cells. Our study investigates the role of TXNIP in erythropoiesis. We observed a rapid and significant increase of TXNIP transcript and protein levels in mouse erythroleukemia cells treated with dimethyl sulfoxide or hexamethylene bisacetamide, inducers of erythroid differentiation. The upregulation of TXNIP was not abrogated by addition of the antioxidant N-acetylcysteine. The increase of TXNIP expression was confirmed in another model of erythroid differentiation, G1E-ER cells, which undergo differentiation upon activation of the GATA1 transcription factor. In addition, we showed that TXNIP levels are induced following inhibition of p38 or c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. We also observed an increase in iron uptake and a decrease in transferrin receptor protein upon TXNIP overexpression, suggesting a role in iron homeostasis. In vivo, flow cytometry analysis of cells from Txnip(-/-) mice revealed a new phenotype of impaired terminal erythropoiesis in the spleen, characterized by a partial block between basophilic and late basophilic/polychromatic erythroblasts. Based on our data, TXNIP emerges as a novel regulator of terminal erythroid differentiation.

Topics: Animals; Anthracenes; Butadienes; Carrier Proteins; Cell Differentiation; Cell Line; Cell Line, Tumor; Erythroblasts; Erythropoiesis; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Heme; Imidazoles; Immunoblotting; Iron; JNK Mitogen-Activated Protein Kinases; Leukemia, Erythroblastic, Acute; Mice, Inbred C57BL; Mice, Knockout; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; Thioredoxins

Regulation of intestinal secretion is important for body fluid homeostasis. We investigated the role of three MAP kinases (MAPKs) as negative regulators in muscarinic cholinoceptor (mAChR)-mediated intestinal secretion in mice. Electrophysiological analyses revealed that mAChR stimulation enhanced intestinal chloride secretion, which was further augmented by the inhibition of JNK but not by that of ERK or p38 with specific inhibitors SP600125, U0126 or SB203580, respectively. Immunoblot analyses in colonic mucosa showed that mAChR stimulation increased MAPKs phosphorylation that was suppressed by the specific inhibitor for each MAPK. This suggests that JNK is a major negative regulator in mAChR-induced intestinal secretion.

Topics: Animals; Anthracenes; Butadienes; Extracellular Signal-Regulated MAP Kinases; Imidazoles; Intestinal Secretions; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Membrane Potentials; Mice; Muscarinic Agonists; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines

TNFα receptors are constitutively overexpressed in tumor cells, correlating to sustain elevated NFκB and monocyte chemotactic protein-1 (MCP-1/CCL2) expression. The elevation of CCL2 evokes aggressive forms of malignant tumors marked by tumor associated macrophage (TAM) recruitment, cell proliferation, invasion and angiogenesis. Previously, we have shown that the organo-sulfur compound diallyl disulfide (DADS) found in garlic (Allium sativum) attenuates TNFα induced CCL2 production in MDA-MB-231 cells. In the current study, we explored the signaling pathways responsible for DADS suppressive effect on TNFα mediated CCL2 release using PCR Arrays, RT-PCR and western blots. The data in this study show that TNFα initiates a rise in NFκB mRNA, which is not reversed by DADS. However, TNFα induced heightened expression of IKKε and phosphorylated ERK. The expression of these proteins corresponds to increased CCL2 release that can be attenuated by DADS. CCL2 induction by TNFα was also lessened by inhibitors of p38 (SB202190) and MEK (U0126) but not JNK (SP 600125), all of which were suppressed by DADS. In conclusion, the obtained results indicate that DADS down regulates TNFα invoked CCL2 production primarily through reduction of IKKε and phosphorylated-ERK, thereby impairing MAPK/ERK, and NFκB pathway signaling. Future research will be required to evaluate the effects of DADS on the function and expression of TNFα surface receptors.

Topics: Allyl Compounds; Anthracenes; Anticarcinogenic Agents; Butadienes; Cell Line, Tumor; Chemokine CCL2; Disulfides; Garlic; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Macrophages; MAP Kinase Kinase 4; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; NF-kappa B p50 Subunit; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Tumor Necrosis Factor-alpha

Naringin, an active flavonoid isolated from citrus fruit extracts, exhibits biological and pharmacological properties, such as antioxidant activity and antidiabetic effect. Mitogen-activated protein kinase (MAPK) signalling pathway has been shown to participate in hyperglycaemia-induced injury. The present study tested the hypothesis that naringin protects against high glucose (HG)-induced injuries by inhibiting MAPK pathway in H9c2 cardiac cells. To examine this, the cells were treated with 35 mM glucose (HG) for 24 hr to establish a HG-induced cardiomyocyte injury model. The cells were pre-treated with 80 μM naringin for 2 hr before exposure to HG. The findings of this study showed that exposure of H9c2 cells to HG for 24 hr markedly induced injuries, as evidenced by a decrease in cell viability, increases in apoptotic cells and reactive oxygen species (ROS) production, as well as dissipation of mitochondrial membrance potential (MMP). These injuries were significantly attenuated by the pre-treatment of cells with either naringin or SB203580 (a selective inhibitor of p38 MAPK) or U0126 (a selective inhibitor of extracellular signal regulated kinase 1/2, ERK1/2) or SP600125 (a selective inhibitor of c-jun N-termanal kinase, JNK) before exposure to HG, respectively. Furthermore, exposure of cells to HG increased the phosphorylation of p38 MAPK, ERK1/2 and JNK. The increased activation of MAPK pathway was ameliorated by pre-treatment with either naringin or N-acetyl-L-cysteine (NAC), a ROS scavenger, which also reduced HG-induced cytotoxicity and apoptosis, leading to increase in cell viability and decrease in apoptotic cells. In conclusion, our findings provide new evidence for the first time that naringin protects against HG-induced injuries by inhibiting the activation of MAPK (p38 MAPK, ERK1/2 and JNK) and oxidative stress in H9c2 cells.

Topics: Acetylcysteine; Animals; Anthracenes; Apoptosis; Butadienes; Cell Line; Cell Survival; Enzyme Inhibitors; Flavanones; Fruit; Glucose; Hyperglycemia; Imidazoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Nitriles; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plant Extracts; Pyridines; Rats; Reactive Oxygen Species

Resistin and endothelin (ET)-1 have been reported to inhibit adipogenesis and regulate adipocyte insulin resistance, respectively. Although both hormones interact with each other, the exact signaling pathway of ET-1 to act on resistin gene expression is still unknown. Using 3T3-L1 adipocytes, we investigated the signaling pathways involved in ET-1-stimulated resistin gene expression. The up-regulation of resistin mRNA expression by ET-1 depends on concentration and timing. The concentration of ET-1 that increased resistin mRNA levels by 100%-250% was approximately 100 nM for a range of 0.25-12 hours of treatment. Treatment with actinomycin D blocked ET-1-increased resistin mRNA levels, suggesting that the effect of ET-1 requires new mRNA synthesis. Treatment with an inhibitor of the ET type-A receptor, such as N-[1-Formyl-N-[N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophyl]-D-tryptophan (BQ610), but not with the ET type-B receptor antagonist N-[(cis-2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine (BQ788), blocked ET-1, increased the levels of resistin mRNA, and phosphorylated levels of downstream signaling molecules, such as ERK1/2, c-Jun N-terminal kinases (JNKs), protein kinase B (AKT), and signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment of specific inhibitors of either ERK1/2 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene [U0126] and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one [PD98059], two inhibitors of MEK1), JNKs (SP600125), phosphatidylinositol 3-kinase/AKT (LY294002 and Wortmannin), or Janus kinase 2 (JAK2)/STAT3 ((E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide, AG490) prevented ET-1-increased levels of resistin mRNA and reduced the ET-1-stimulated phosphorylation of ERK1/2, JNKs, AKT, and STAT3, respectively. However, the p38 kinase antagonist 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580) did not alter the effect of ET-1. These results imply that ET type-A receptor, ERK1/2, JNKs, AKT, and JAK2, but not ET type-B receptor or p38, are necessary for the ET-1 stimulation of resistin gene expression. In vivo observations that ET-1 increased resistin mRNA and protein levels in sc and epididymal adipose tissues support the in vitro findings.

Topics: 3T3-L1 Cells; Adipose Tissue; Androstadienes; Animals; Anthracenes; Butadienes; Chromones; Dactinomycin; Endothelin-1; Flavonoids; Gene Expression Profiling; Gene Expression Regulation; Male; MAP Kinase Kinase 4; Mice; Mice, Inbred C57BL; Morpholines; Nitriles; Oligopeptides; Piperidines; Proto-Oncogene Proteins c-akt; Resistin; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Wortmannin

Conditioned mediums (CMs) from glioma cells U87, GBM-8401, and C6 significantly induced iNOS protein and NO production by microglial cells BV-2 but without altering the cell viability or cell-cycle progression of BV2 microglia. Significant increases in intracellular peroxide by U87-CM and C6-CM were detected by a DCHF-DA assay, and vitamin (Vit) C and N-acetyl cysteine (NAC)-reduced intracellular peroxide levels elicited by CMs lead to inhibition of iNOS/NO production The extracellular signal-regulated kinase (ERK) inhibitor, U0126, and c-Jun N-terminal kinase (JNK) inhibitor, SP600125, suppressed U87-CM- and C6-CM-induced iNOS/NO production by respectively blocking phosphorylated ERK (pERK) and JNK (pJNK) protein expressions stimulated by U87-CM and C6-CM. Increased migration of U87 and C6 glioma cells by a co-culture with BV-2 microglial cells or adding the nitric oxide (NO) donor, sodium nitroprusside (SNP) was observed, and that was blocked by adding an NO synthase (NOS) inhibitor, N-nitro L-arginine methyl ester (NAME). Contributions of ROS, pERK, and pJNK to the migration of glioma cells was further demonstrated in a transwell coculture system of U87 and C6 gliomas with BV-2 microglial cells. Furthermore, expressions of tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 messenger (m)RNA in U87 and C6 cells were detected by an RT-PCR, and TNF-α and MCP-1 induced iNOS protein expression in time- and concentration-dependent manners. Neutralization of TNF-α or MCP-1 in U87-CM and C6-CM using a TNF-α or MCP-1 antibody inhibited iNOS protein expression, and increased intracellular peroxide by TNF-α or MCP-1 was identified in BV-2 cells. The reciprocal activation of glioma cells and microglia via ROS-dependent iNOS/NO elevation at least partially mediated by TNF-α and MCP-1 is elucidated.

Topics: Anthracenes; Butadienes; Cell Line, Tumor; Cell Survival; Chemokine CCL2; Coculture Techniques; Culture Media, Conditioned; Gene Expression Regulation, Neoplastic; Glioma; Humans; JNK Mitogen-Activated Protein Kinases; Microglia; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Nitriles; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Biodentine (Septodont, Saint-Maur-des-Fossès, France), a new tricalcium silicate cement formulation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and calcium-/calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation.. hDPCs obtained from impacted third molars were incubated with Biodentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase polymerase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-κB (pyrrolidine dithiocarbamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay.. Biodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal kinase (SP600125) significantly decreased the Biodentine-induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-κB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization.. Biodentine is a bioactive and biocompatible material capable of inducing odontoblast differentiation of hDPSCs. Our results indicate that this induction is regulated via MAPK and CaMKII pathways.

Topics: Adolescent; Adult; Alkaline Phosphatase; Anthracenes; Benzylamines; Butadienes; Calcium Compounds; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Culture Techniques; Cell Differentiation; Dental Pulp; Extracellular Matrix Proteins; Humans; Imidazoles; Integrin-Binding Sialoprotein; MAP Kinase Signaling System; NF-kappa B; Nitriles; Odontoblasts; Osteocalcin; Phosphoproteins; Protein Kinase Inhibitors; Pulp Capping and Pulpectomy Agents; Pyridines; Pyrrolidines; Sialoglycoproteins; Signal Transduction; Silicates; Stem Cells; Sulfonamides; Thiocarbamates; Young Adult

Hepatocyte growth factor (HGF) is a protective factor in myocardial injury, but its mechanisms of action have not yet been fully elucidated. Nexilin, which locates specifically to the Z-disc, is a novel Z-disc protein that enables the Z-discs to persistently withstand the extreme mechanical forces generated during muscle contraction. Therefore, we investigated the role of HGF in modulating nexilin expression in hypoxia-reoxygenation (H/R)-treated cardiomyocytes. We cultured neonatal cardiomyocytes and treated them with HGF. The mRNA and protein levels of nexilin were determined by RT-PCR and Western blotting. H/R treatment decreased nexilin mRNA expression and nexilin protein levels in cardiomyocytes. Furthermore, treatment with HGF upregulated nexilin expression and the JNK inhibitor SP600125 partly inhibited HGF-induced nexilin upregulation. In conclusion, our results suggest that ischemia-reperfusion injury may downregulate nexilin expression in cardiomyocytes, and HGF may exert its protective role during myocardial ischemic injury through upregulation of nexilin expression in cardiomyocytes.

Topics: Animals; Animals, Newborn; Anthracenes; Butadienes; Cell Hypoxia; Flavonoids; Gene Expression Regulation; Heart Ventricles; Hepatocyte Growth Factor; JNK Mitogen-Activated Protein Kinases; Microfilament Proteins; Myocytes, Cardiac; Nitriles; Oxygen; Primary Cell Culture; Protein Kinase Inhibitors; Rats; RNA, Messenger; Signal Transduction

The aim of the present study was to investigate the effects of lipopolysaccharide (LPS) on the migration and adhesion of human dental pulp stem cells (hDPSCs) and the associated intracellular signalling pathways.. hDPSCs obtained from impacted third molars were exposed to LPS and in vitro cell adhesion and migration were evaluated. The effects of LPS on gene expression of adhesion molecules and chemotactic factors were investigated using quantitative real-time reverse-transcriptase polymerase chain (qRT-PCR). The potential involvement of nuclear factor NF-kappa-B (NF-κB) or mitogen-activated protein kinase (MAPK) signalling pathways in the migration and adhesion of hDPSCs induced by LPS was assessed using a transwell cell migration assay and qRT-PCR.. LPS promoted the adhesion of hDPSCs at 1μg/mL and 10μg/mL concentrations, 1μg/mL LPS showing the greater effect. Transwell cell migration assay demonstrated that LPS increased migration of hDPSCs at 1μg/mL concentration while decreasing it significantly at 10μg/mL. The mRNA expressions of adhesion molecules and chemotactic factors were enhanced significantly after stimulation with 1μg/mL LPS. Specific inhibitors for NF-κB and extracellular signal regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and P38, markedly antagonised LPS-induced adhesion and migration of hDPSCs and also significantly abrogated LPS-induced up-regulation of adhesion molecules and chemotactic factors. In addition, specific inhibitors of SDF-1/CXCR4, AMD3100 significantly diminished LPS-induced migration of hDPSCs.. LPS at specific concentrations can promote cell adhesion and migration in hDPSCs via the NF-κB and MAPK pathways by up-regulating the expression of adhesion molecules and chemotactic factors.. LPS may influence pulp healing through enhancing the adhesion and migration of human dental pulp stem cells when it enters into pulp during pulp exposure or deep caries.

Topics: Adolescent; Adult; Anthracenes; Benzylamines; Butadienes; Cell Adhesion; Cell Adhesion Molecules; Cell Movement; Cells, Cultured; Chemokine CXCL12; Chemotactic Factors; Cyclams; Dental Pulp; Escherichia coli; Extracellular Signal-Regulated MAP Kinases; Heterocyclic Compounds; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; MAP Kinase Signaling System; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Pyrrolidines; Receptors, CXCR4; Signal Transduction; Stem Cells; Thiocarbamates; Young Adult

Mesenchymal stem cells (MSCs) are able to home and migrate into damaged tissues and are thus, considered an optimal therapeutic strategy for clinical use. We previously demonstrated that higher shear stress (>2 Pa) hindered human MSC (hMSC) migration, whereas lower shear stress (0.2 Pa) induced cell migration through mitogen-activated protein kinase (MAPK) pathways. Here the mechanisms underlying shear stress-induced hMSC migration have been studied further. An MSC monolayer was mechanically wounded and subsequently exposed to low-level shear stress of 0.2 Pa. Image analysis was performed to quantify cell migration speeds under both flow and static conditions. hMSCs along both upstream- and downstream edges of the wound migrated at a similar speed to cover the wounded area under static conditions, whereas shear stress induced cells along the downstream edge of the wound to migrate significantly faster than those along the upstream edge. We also found that shear stress upregulated the secretion of stromal-derived factor-1 (SDF-1), which stimulated its receptor CXCR4 expression in hMSCs until the cells covered the wounded area. A CXCR4 antagonist repressed both cell migration and activation of c-Jun N-terminal kinase (JNK) and p38 MAPK but did not affect extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. When MAPK activation in upstream- and downstream hMSCs was evaluated separately, ERK1/2 was activated earlier in downstream than in upstream cells. These results indicate that the SDF-1/CXCR4 axis mediates shear stress-induced hMSC migration through JNK and p38 MAPK pathways and that the difference in migration speeds between upstream- and downstream cells may be due to ERK1/2 activation.

Topics: Anthracenes; Benzylamines; Butadienes; Cell Movement; Cell- and Tissue-Based Therapy; Cells, Cultured; Chemokine CXCL12; Cyclams; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Heterocyclic Compounds; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mesenchymal Stem Cells; Mitogen-Activated Protein Kinases; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Receptors, CXCR4; Stress, Mechanical; Wound Healing

In this study, BRL 3A cells were treated with different Cd concentrations (0, 10, 20, and 40 μmol/L) for 12 h and preincubated with or without N-acetyl-L-cysteine (NAC) (2 mmol/L) for 30 min, and cells were treated with Cd (0 and 20 μmol/L), pretreated with p38 inhibitor (SB203580), JNK (c-Jun NH2-terminal kinases) inhibitor (SP600125), and extracellular signal-regulated kinase (ERK) inhibitor (U0126) for 30 min, and then treated with 20 μmol/L Cd for 12 h. Cd decreased cell viability, SOD, and GSH-Px activity in a concentration-dependent manner. Increased MDA level, ROS generation, nuclear condensation, shrinkage, and fragmentation in cell morphology were inhibited by NAC. Cd-induced apoptosis was attenuated by pretreatment with SB203580, SP600125, and U0126. The results of western blot showed that NAC preincubation affected Cd-activated MAPK pathways, p38 and ERK phosphorylation. Cd treatment elevated the mRNA levels of Bax and decreased the mRNA levels of Bcl-2, respectively. The same effect was found in their protein expression levels. These results suggest that oxidative stress and MAPK pathways participate in Cd-induced apoptosis and that the balance between pro- and antiapoptotic genes (Bax and Bcl-2) is important in Cd-induced apoptosis.

Topics: Acetylcysteine; Animals; Anthracenes; Apoptosis; Butadienes; Cadmium; Cell Line; Extracellular Signal-Regulated MAP Kinases; Glutathione Peroxidase; Imidazoles; JNK Mitogen-Activated Protein Kinases; Malondialdehyde; Mitogen-Activated Protein Kinases; Nitriles; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Rats; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase

Mineral trioxide aggregate (MTA) can induce differentiation of the dental pulp cells into odontoblast-like cells and generate a dentin-like mineral structure. The mechanisms underlying MTA-induced odontoblastic differentiation in human dental pulp cells (HDPCs) are not completely understood. The purpose of this study was to evaluate the effect of nifedipine as calcium channel blocker on MTA-induced odontoblastic differentiation in HDPCs.. HDPCs extracted from maxillary supernumerary incisors and third molars were directly cultured on MTA with or without nifedipine in the culture medium. Cell growth and expression of odontoblastic differentiation markers were determined by using methyl-thiazol-diphenyl-tetrazolium assay and reverse transcription-polymerase chain reaction analysis, respectively. Phosphorylation of mitogen-activated protein kinase was measured by Western blotting, and calcium deposition was assessed by using alizarin red S staining.. MTA at a concentration of 1 mg/mL significantly up-regulated the expression of dentin sialophosphoprotein and dentin matrix protein-1 and enhanced mineralized nodule formation. However, nifedipine attenuated the MTA-induced odontoblastic differentiation in HDPCs. In addition, MTA-induced mineralization was blocked by inhibition of extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) by using U0126, SB203580, and SP600125, respectively. Furthermore, phosphorylation of ERK and JNK in response to MTA was inhibited when the medium was supplemented with nifedipine.. This study showed that calcium ions released from MTA play an important role in odontoblastic differentiation of HDPCs via modulation of ERK and JNK activation.

Topics: Adult; Aluminum Compounds; Anthracenes; Butadienes; Calcium; Calcium Channel Blockers; Calcium Compounds; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cells, Cultured; Culture Media; Dental Pulp; Drug Combinations; Extracellular Matrix Proteins; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Nifedipine; Nitriles; Odontoblasts; Oxides; p38 Mitogen-Activated Protein Kinases; Phosphoproteins; Pyridines; Root Canal Filling Materials; Sialoglycoproteins; Silicates; Tooth Calcification

More than 25% of patients diagnosed with endometrial carcinoma have an invasive primary cancer accompanied by metastases. Gonadotropin-releasing hormone (GnRH) plays an important role in reproduction. In mammals, expression of GnRH-II is higher than GnRH-I in reproductive tissues. Here, we examined the effect of a GnRH-II agonist on the motility of endometrial cancer cells and its mechanism of action in endometrial cancer therapy.. Immunoblotting and immunohistochemistry (IHC) were used to determine the expression of the GnRH-I receptor protein in human endometrial cancer. The activity of MMP-2 in the conditioned medium was determined by gelatin zymography. Cell motility was assessed by invasion and migration assay. GnRH-I receptor si-RNA was applied to knockdown GnRH-I receptor.. The GnRH-I receptor was expressed in the endometrial cancer cells. The GnRH-II agonist promoted cell motility in a dose-dependent manner. The GnRH-II agonist induced the phosphorylation of ERK1/2 and JNK, and the phosphorylation was abolished by ERK1/2 inhibitor (U0126) and the JNK inhibitor (SP600125). Cell motility promoted by GnRH-II agonist was suppressed in cells that were pretreated with U0126 and SP600125. Moreover, U0126 and SP600125 abolished the GnRH-II agonist-induced activation of MMP-2. The inhibition of MMP-2 with MMP-2 inhibitor (OA-Hy) suppressed the increase in cell motility in response to the GnRH-II agonist. Enhanced cell motility mediated by GnRH-II agonist was also suppressed by the knockdown of the endogenous GnRH-I receptor using siRNA.. Our study indicates that GnRH-II agonist promoted cell motility of endometrial cancer cells through the GnRH-I receptor via the phosphorylation of ERK1/2 and JNK, and the subsequent, MAPK-dependent activation of MMP-2. Our findings represent a new concept regarding the mechanism of GnRH-II-induced cell motility in endometrial cancer cells and suggest the possibility of exploring GnRH-II as a potential therapeutic target for the treatment of human endometrial cancer.

Topics: Anthracenes; Butadienes; Carcinoma; Cell Line, Tumor; Cell Movement; Endometrial Neoplasms; Enzyme Activation; Female; Gene Knockdown Techniques; Gonadotropin-Releasing Hormone; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Nitriles; Phosphorylation; Receptors, LHRH

Claudin-4 is exclusively localized in the tight collecting ducts in the renal tubule. We examined what molecular mechanism is involved in the regulation of claudin-4 expression. In Madin-Darby canine kidney cells, hyperosmolarity increased the expression level of claudin-4 and the production of reactive oxygen species, which were inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, and manganese (III) tetrakis (4-benzoic acid)porphyrin (MnTBAP), a scavenger of H2O2. Both hyperosmolarity and H2O2 increased p-ERK1/2 and p-JNK, which were inhibited by U0126, a MEK inhibitor, and SP600125, a JNK inhibitor, respectively. Immunoprecipitation assay showed that hyperosmolarity increased the association of nuclear Sp1 with c-Jun, which was inhibited by U0126 and SP600125. In mouse inner medullary collecting duct cells and rat kidney slices, hyperosmolarity increased the expression level of claudin-4, which was inhibited by DPI, MnTBAP, U0126, and SP600125. Hyperosmolarity increased luciferase reporter activity of claudin-4, which was inhibited by U0126, SP600125, Sp1 siRNA, and c-Jun siRNA. The activity was inhibited by the mutation in the Sp1 binding site. Chromatin immunoprecipitation assay and avidin-biotin conjugated DNA assay showed that Sp1 and c-Jun are associated with the Sp1 binding site. These results suggest that hyperosmolarity increases nuclear Sp1/c-Jun complex and the association of the complex with the Sp1 binding site, resulting in the segment-specific expression of claudin-4 in the kidney.

Topics: Animals; Anthracenes; Base Sequence; Butadienes; Cell Nucleus; Claudin-4; Cycloheximide; Dogs; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Free Radical Scavengers; Hydrogen Peroxide; Hypertonic Solutions; JNK Mitogen-Activated Protein Kinases; Kidney Tubules, Collecting; Madin Darby Canine Kidney Cells; Models, Biological; Molecular Sequence Data; NADPH Oxidases; Nitriles; Osmolar Concentration; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Sp1 Transcription Factor; Up-Regulation

Connexin 43 (Cx43) is a major structural protein found in the gap junctions of the ventricular myocardium and a major determinant of its electrical properties. The effects of matrix metalloproteinases (MMPs), the mitogen-activated protein kinase (MAPK) signaling pathway, transcription factor NF-kB, and activator protein-1 (AP-1)/c-Jun on the regulation of Cx43 gene expression in H9c2 cardiomyocytes were assessed. The MAPK signaling pathway (MEK/ERK1/2 and PI3K) and transcription factors NF-kB and AP-1/c-Jun were inhibited, then Cx43 expression was assessed using Western blot analysis, and MMP-9 activity was assessed using gelatin zymography. Hypoxia decreased the Cx43 protein level by approximately 30-50 %. Doxycycline (10 μg/mL), an inhibitor of MMP, markedly attenuated the hypoxia-induced downregulation of Cx43 protein expression at 6 h. The hypoxia-induced decrease in Cx43 protein expression was significantly reversed by U0126 (10 μM), a MEK/ERK1/2 inhibitor, at 6 and 12 h; LY294002 (30 μM), a PI3K inhibitor, downregulated Cx43 expression. Hypoxia-induced MMP-9 activation was inhibited by treatment with LY294002, U0126, and, most especially, U0126. JSH-23 (30 μM), an NF-kB inhibitor, and SP600125 (10 μM), an AP-1/c-Jun inhibitor, attenuated the loss of Cx43. These results suggest that MAPK signaling and the activities NF-kB and MMPs play an important roles in the regulation of Cx43 expression.

Topics: Animals; Anthracenes; Butadienes; Cell Hypoxia; Cell Survival; Cells, Cultured; Chromones; Connexin 43; Down-Regulation; Doxycycline; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Morpholines; Myocytes, Cardiac; NF-kappa B; Nitriles; Phenylenediamines; Phosphoinositide-3 Kinase Inhibitors; Promoter Regions, Genetic; Rats; Transcription Factor AP-1

To examine CD99 expression and its functional role in ICAM-1 induction in human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGECs) by activating cells with anti-CD99 monoclonal antibody, MT99/3.. Engagement of CD99 with agonistic antibodies has been shown to regulate immune responses, cell adhesion and migration, and cell death in several studies. Particularly, this engagement results in transendothelial migration of leukocytes mediated by intercellular adhesion molecule-1 (ICAM-1) induction in endothelial cells.. Total mRNA and protein were isolated from HGFs and HGECs for analyses of CD99 and ICAM-1 expression. Surface expression of CD99 and ICAM-1 was analysed by flow cytometry, and the detection of soluble ICAM-1 was assayed by immunoprecipitation and ELISA.. CD99 surface expression was constitutive on HGFs to a greater extent than that on HGECs. CD99 ligation with MT99/3 induced ICAM-1 mRNA expression in HGFs, but not in HGECs. Interestingly, CD99 ligation led to an increased level of soluble ICAM-1 detected in culture supernatant, whereas interleukin-1β (IL-1β) treatment induced expression of membrane-bound ICAM-1. Furthermore, ICAM-1 induction by CD99 engagement was demonstrated to involve the activation of the p50 subunit of nuclear factor-kappaB (NF-κB), extracellular signal-regulated kinase, and p46 c-Jun N-terminal kinase that differed from that by IL-1β treatment.. Our study has shown the involvement of CD99 ligation in the up-regulation of ICAM-1 expression and its secretion in gingival fibroblasts, which may be essential for better understanding of the pathogenesis of periodontal disease.

Topics: 12E7 Antigen; Anthracenes; Antibodies, Monoclonal; Antigens, CD; Butadienes; Cell Adhesion Molecules; Cell Movement; Cells, Cultured; Culture Media, Conditioned; Endothelial Cells; Enzyme Inhibitors; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Flow Cytometry; Gingiva; Humans; Imidazoles; Intercellular Adhesion Molecule-1; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Leukocytes; Leupeptins; MAP Kinase Signaling System; NF-kappa B; NF-kappa B p50 Subunit; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines

Human dermal fibroblasts (HDFs) regulate wound-healing processes in human skin, including the regeneration of skin sensory fibres, in response to various mechanical stimuli. Because nerve growth factor (NGF) has an essential role in sensory regeneration, we evaluated the possible association of NGF with mechanical stimulus-dependent cellular responses in HDFs. A cyclic tensile stimulus increased both NGF and transforming growth factor (TGF) β2 production, yet with different gene transcription and signal desensitization profiles. Neutralizing TGFβ with antibodies did not affect the tension-induced NGF upregulation, with significant inhibition of endogenous TGFβ2 transcription. The treatment with LY294002, SP600125 or U0126 hindered the tension-induced TGFβ2 upregulation, although the increase in NGF was regulated only by SP600125 or U0126, indicating the involvement of three signalling kinase pathways in the upregulation of TGFβ2. However, the upregulation of NGF was shown to be independent of PI3K, demonstrating the independent regulation of tension-induced NGF and TGFβ production in HDFs.

Topics: Anthracenes; Antibodies, Neutralizing; Butadienes; Chromones; Fibroblasts; Humans; Morpholines; Nerve Growth Factor; Nitriles; RNA, Messenger; Signal Transduction; Skin; Stress, Mechanical; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Up-Regulation

The abnormal proliferation and migration of vascular smooth muscle cell (VSMC) contributes importantly to the pathogenesis of atherosclerosis and restenosis. Here, we investigated the effects of eupatolide (EuTL), a sesquiterpene lactone isolated from the medicinal plant Inula britannica, on platelet-derived growth factor (PDGF)-induced proliferation and migration of primary rat aortic smooth muscle cells (RASMCs), as well as its underlying mechanisms. EuTL remarkably inhibited PDGF-induced proliferation and migration of RASMCs. Treatment of RASMCs with EuTL induced both protein and mRNA expression of heme oxygenase-1 (HO-1). SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor) and LY294002 (a PI3K inhibitor) did not suppress EuTL-induced HO-1 expression; however, N-acetylcysteine (NAC, an antioxidant) blocked EuTL-induced HO-1 expression. Moreover, treatment of RASMCs with EuTL increased reactive oxygen species (ROS) accumulation and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2); however, this translocation was also inhibited by NAC. NAC or inhibition of HO-1 significantly attenuated the inhibitory effects of EuTL on PDGF-induced proliferation and migration of RASMCs. Taken together, these findings suggest that EuTL could suppress PDGF-induced proliferation and migration of VSMCs through HO-1 induction via ROS-Nrf2 pathway and may be a potential HO-1 inducer for preventing or treating vascular diseases.

Topics: Acetylcysteine; Animals; Anthracenes; Aorta; Butadienes; Cell Movement; Cell Proliferation; Cells, Cultured; Chromones; Enzyme Inhibitors; Heme Oxygenase (Decyclizing); Imidazoles; Inula; Male; Morpholines; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-E2-Related Factor 2; Nitriles; Platelet-Derived Growth Factor; Pyridines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sesquiterpenes

To determine whether the long pentraxin 3 (PTX3) is expressed in human retinal pigment epithelial cells and is induced by inflammatory cytokines, interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), expression of PTX3 was investigated in the human retinal pigment epithelial cell line, ARPE-19 cells.. In ARPE-19 cells, we first analyzed PTX3 production in the presence or absence of inflammatory cytokines, IL-1β, TNF-α, and IFN-γ, dose- and time-dependently using enzyme-linked immunosorbent assay. Protein and mRNA expression of PTX3 was measured with western blotting analysis and real-time reverse transcription-polymerase chain reaction. Specific inhibitors were used to determine the signaling pathways of inflammatory cytokine-induced PTX3 expression.. In this study, production of PTX3 was induced by IL-1β and TNF-α dose- and time-dependently, but not by IFN-γ in ARPE-19 cells. Protein and mRNA expression of PTX3 was significantly upregulated in the presence of IL-1β and TNF-α. Furthermore, pretreatment with extracellular signal-regulated kinase1/2 and nuclear factor kappa-light-chain-enhancer of activated B cells specific inhibitor abolished IL-1β and TNF-α-induced PTX3 production, but the other inhibitors had no effect.. These results suggested that human retinal pigment epithelial cells may be a major source of PTX3 production in the presence of proinflammatory cytokines, IL-1β and TNF-α, and could be an important mediator for host defense and inflammatory response in the retina. The importance of the mitogen-activated protein kinase/extracellular signal-regulated kinase1/2 and nuclear factor kappa-light-chain-enhancer of activated B cells pathways for regulated PTX3 expression may be a potential target for PTX3 regulation in the retina.

Topics: Anthracenes; Butadienes; C-Reactive Protein; Cell Line; Cytokines; Gene Expression; Humans; Imidazoles; Inflammation Mediators; Interferon-gamma; Interleukin-1beta; MAP Kinase Signaling System; NF-kappa B; Nitriles; Pyridines; Retinal Pigment Epithelium; RNA, Messenger; Serum Amyloid P-Component; Signal Transduction; Sulfones; Tumor Necrosis Factor-alpha

The aim of this study was to investigate whether norepinephrine (NE) could regulate macrophage production of tumor necrosis factor alpha (TNF-α) by influencing the phosphorylation of mitogen-activated protein kinases (MAPKs). Primary macrophages from male BALB/c mice were applied to explore the mechanism by which NE influences the the secretion of TNF-α when macrophages were activated by lipopolysaccharides (LPS). We found that NE could increase crophage production of TNF-α when macrophages were activated by LPS, and this effect could be inhibited by α adrenergic antagonist phentolamine. Also, NE could increase the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), and p38, through α receptor. Furthermore, JNK inhibitor SP600125, ERK inhibitor U0126, and p38 inhibitor SB203580 could all partially counteract NE's effect on the phosphorylation of MAPKs, as well as TNF-α production by macrophages. This study revealed that as macrophages were activated by LPS, NE promoted the secretion of inflammatory factors by increasing the phosphorylation of MAPKs through an α receptor-dependent pathway. Our results provide the evidence of a relationship between stress and diseases, as well as the mechanism by which stress induces or affects the inflammation-related diseases.

Topics: Adrenergic alpha-Antagonists; Animals; Anthracenes; Butadienes; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Macrophage Activation; Macrophages; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Nitriles; Norepinephrine; p38 Mitogen-Activated Protein Kinases; Phentolamine; Phosphorylation; Pyridines; Receptors, Adrenergic, alpha; Tumor Necrosis Factor-alpha

Age-related macular degeneration (AMD) is the predominant cause of irreversible blindness in the elderly population. Despite intensive basic and clinical research, its pathogenesis remains unclear. However, evidence suggests that immunological and inflammatory factors contribute to the pathogenesis of AMD. A newly identified cytokine, IL-33, appears to be an important pro-inflammatory cytokine promoting tissue inflammation. In this study, IL-33 was increased through amyloid-beta(1-40) (Aβ(1-40)) stimulation and regulated inflammatory cytokines including IL-6, IL-8, IL-1β, and TNF-α secretion using different signaling pathways in retinal pigment epithelium (RPE) cells. Furthermore, ST2L, the important component of the IL-33 receptor, was significantly increased following recombinant human IL-33 stimulation in RPE cells. These findings suggest that IL-33-mediated inflammatory responses in RPE cells are involved in the pathogenesis of AMD. Greater understanding of the inflammatory effect of IL-33 and its role in RPE cells should aid the development of future clinical therapeutics and enable novel pharmacological approaches towards the prevention of AMD.

Topics: Amyloid beta-Peptides; Anthracenes; Butadienes; Cell Line; Humans; Imidazoles; Inflammation; Interleukin-1 Receptor-Like 1 Protein; Interleukin-1beta; Interleukin-33; Interleukin-6; Interleukin-8; Interleukins; JNK Mitogen-Activated Protein Kinases; Macular Degeneration; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Nitriles; Peptide Fragments; Pyridines; Receptors, Cell Surface; Retinal Pigment Epithelium; Sulfones; Tumor Necrosis Factor-alpha

Although it has been suggested that smad7 blocks downstream signaling of TGF-β, the role of smad7 in the EGF signaling pathway has not been fully elucidated. We determined the effect of smad7 on EGF-induced MMP-9 expression in SKBR3 breast cancer cells. The expression of smad7 and MMP-9 was increased by EGF or TGF-β1, respectively, and further increased by EGF and TGF-β1 co-treatment. EGF induced the phosphorylation of EGFR, smad3, ERK, and JNK, and MMP-9 expression was decreased by the EGFR inhibitor, AG1478. In addition, EGF-induced MMP-9 expression was inhibited by UO126 (a MEK1/2 inhibitor) or SIS3 (a smad3 inhibitor), but not by SP600125 (a JNK inhibitor). Interestingly, EGF-induced smad3 phosphorylation was completely blocked by smad7 over-expression, but not the phosphorylation of ERK and JNK. EGF- or TGF-β1-induced MMP-9 expression was completely decreased by adenoviral-smad7 (Ad-smad7) over-expression. We also investigated the role of smad3 on EGF-induced MMP-9 expression and showed that EGF-induced MMP-9 expression was decreased by smad3 siRNA transfection, whereas EGF-induced MMP-9 expression was further increased by smad3 over-expression, as expected. This study showed that EGF-induced smad3 phosphorylation mediates the induction of MMP-9, whereas smad7 inhibits TGF-β1 as well as the EGF signaling pathway in SKBR3 cells.

Topics: Anthracenes; Breast Neoplasms; Butadienes; Cell Line, Tumor; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Matrix Metalloproteinase 9; Nitriles; Phosphorylation; RNA, Messenger; Signal Transduction; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta1

Prostaglandin E(2) (PGE(2)) is a bioactive prostanoid implicated in the inflammatory processes of acute lung injury/acute respiratory distress syndrome. This study investigated whether PGE(2) can induce production of interleukin (IL)-8, the major chemokine for neutrophil activation, from human pulmonary microvascular endothelial cells (HPMVECs). PGE(2) significantly enhanced IL-8 protein production with increases in IL-8 mRNA expression and intracellular cAMP levels. HPMVECs expressed only EP4 receptor mRNA. The PGE(2) effects were mimicked by a selective EP4 receptor agonist, ONO-AE1-329, and inhibited by a selective EP4 receptor antagonist, ONO-AE3-208, or a protein kinase A inhibitor, Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamine salt. The specific agonist for EP1, EP2, or EP3 receptor did not induce IL-8 production. PGE(2)-induced IL-8 production was accompanied by p38 phosphorylation and was significantly inhibited by a p38 inhibitor, SB-203580, but not by an ERK1/2 inhibitor, U-0126, or a JNK inhibitor, SP-600125. Additionally, PGE(2) increased cyclooxygenase-2 expression with no change in constitutive cyclooxygenase-1 expression, suggesting possible involvement of an autocrine or paracrine manner. In conclusion, PGE(2) enhances IL-8 production via EP4 receptor coupled to G(s) protein in HPMVECs. Activation of the cAMP/protein kinase A pathway, followed by p38 activation, is essential for these mechanisms. Because neutrophils play a critical role in the inflammation of acute lung injury/acute respiratory distress syndrome, IL-8 released from the pulmonary microvasculature in response to PGE(2) may contribute to pathophysiology of this disease.

Topics: Acute Lung Injury; Anthracenes; Butadienes; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Endothelial Cells; Humans; Imidazoles; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Lung; MAP Kinase Signaling System; Methyl Ethers; Microvessels; Naphthalenes; Neutrophils; Nitriles; p38 Mitogen-Activated Protein Kinases; Phenylbutyrates; Pyridines; Receptors, Prostaglandin E, EP4 Subtype; Respiratory Distress Syndrome; RNA, Messenger; Thionucleotides

Lipopolysaccharide (LPS) has been shown to regulate the function of odontoblasts. However, the molecular mechanisms of the effect of LPS on odontoblasts are poorly understood. Decorin (DCN), one of the major matrix proteoglycans, is known to affect the mineralization of teeth. In this study, we investigated whether LPS can regulate the expression of DCN in odontoblasts and determined the intracellular signaling pathways triggered by LPS.. The DCN messenger RNA and protein expression changes in mouse odontoblast-lineage cells (OLCs) were detected by real-time polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). Whether TLR4, myeloid differentiating factor 88 (MyD88), nuclear factor-kappa B (NF-κB), or mitogen-activated protein kinase (MAPK) pathways were involved in the LPS-induced DCN expression was determined by examined real-time PCR, ELISA, and luciferase activity assay. The activation of extracellular signal-regulated kinase (ERK), p38, and JNK in OLCs was measured by Western blot analysis.. We found that the mouse OLCs expressed DCN. DCN messenger RNA was rapidly induced by LPS in a time- and dose-dependent manner. Pretreatment with a MyD88 inhibitory peptide, a TLR4 antibody, or a specific inhibitor for NF-κB or I Kappa B alpha (IκBα) significantly inhibited LPS-induced DCN expression. Moreover, the LPS-mediated increase in κB-luciferase activity in OLCs was suppressed by the overexpression of dominant negative mutants of TLR4, MyD88, and IκBα but not by a dominant negative mutant of TLR2. In addition, LPS stimulation activated the ERK, p38, and JNK MAPK pathways. The pretreatment of OLCs with specific inhibitors of the ERK, p38, and JNK MAPK pathways markedly offset the LPS-induced up-regulation of DCN expression.. Our results show that LPS stimulation can up-regulate the gene expression of DCN via the TLR4, MyD88, NF-κB, and MAPK pathways in odontoblast cells.

Topics: Animals; Anthracenes; Blotting, Western; Butadienes; Cell Line; Decorin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; I-kappa B Kinase; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Luciferases; Luminescent Agents; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Myeloid Differentiation Factor 88; NF-kappa B p50 Subunit; Nitriles; Odontoblasts; p38 Mitogen-Activated Protein Kinases; Pyridines; Real-Time Polymerase Chain Reaction; Time Factors; Toll-Like Receptor 2; Toll-Like Receptor 4; Up-Regulation

Dihydropyridine calcium channel blockers (CCBs) are more effective in reducing carotid intima-media thickness (IMT) than other classes of antihypertensive drugs due to their vascular effects. However, the mechanism remains to be elucidated.. Ox-LDL induced HUVSMCs proliferation in a time- and dose-dependent manner. When pretreated with three CCBs before 50 μg/ml ox-LDL stimulation, 30 μM lacidipine and 3 μM amlodipine exhibited 27% and 18% decrease of pro-proliferative effect induced by ox-LDL, whereas (S-)-amlodipine did not have any anti-proliferative effect. 30 μM lacidipine inhibited about two-thirds of the ox-LDL induced ROS production in HUVSMCs, whereas amlodipine and (S-)-amlodipine did not have influence on ROS production. The MAPKs pathway inhibitors inhibited the ox-LDL induced proliferation of HUVSMCs.. Our study has demonstrated that lipophilic CCBs, such as lacidipine may inhibit ox-LDL induced proliferation and oxidative stress of VSMCs, and that the ROS-MAPKs pathway might be involved in the mechanism.

Topics: Amlodipine; Anthracenes; Butadienes; Calcium Channel Blockers; Cell Proliferation; Cells, Cultured; Dihydropyridines; Dose-Response Relationship, Drug; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lipoproteins, LDL; MAP Kinase Signaling System; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitriles; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Pyridines; Reactive Oxygen Species; Time Factors

Abnormal vascular smooth muscle cells proliferation is the pathophysiological basis of cardiovascular diseases, such as hypertension, atherosclerosis, and restenosis after angioplasty. Angiotensin II can induce abnormal proliferation of vascular smooth muscle cells, but the molecular mechanisms of this process remain unclear. Here, we explored the role and molecular mechanism of monocyte chemotactic protein-1, which mediated angiotensin II-induced proliferation of rat aortic smooth muscle cells. 1,000 nM angiotensin II could stimulate rat aortic smooth muscle cells' proliferation by angiotensin II type 1 receptor (AT(1)R). Simultaneously, angiotensin II increased monocyte chemotactic protein-1 expression and secretion in a dose-and time-dependent manner through activation of its receptor AT(1)R. Then, monocyte chemotactic protein-1 contributed to angiotensin II-induced cells proliferation by CCR2. Furthermore, we found that intracellular ERK and JNK signaling molecules were implicated in angiotensin II-stimulated monocyte chemotactic protein-1 expression and proliferation mediated by monocyte chemotactic protein-1. These results contribute to a better understanding effect on angiotensin II-induced proliferation of rat smooth muscle cells.

Topics: Angiotensin II; Animals; Anthracenes; Aorta, Thoracic; Benzoxazines; Butadienes; Cell Proliferation; Cells, Cultured; Chemokine CCL2; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitriles; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptors, CCR2

several diseases such as sepsis can affect the ileum. Lipopolysaccharide (LPS), an endotoxin present in the cell wall of gram negative bacteria, is a causative agent of sepsis.. the aims of this study were: a) to investigate the role of mitogen activated protein kinases (MAPKs) in the effect of LPS on the acetylcholine-induced contractions of rabbit ileum; and b) to study the localization of MAPKs in the ileum.. ileal contractility was studied in an organ bath and MAPKs were localized by immunohistochemistry.. acetylcholine-induced contractions decreased with LPS. SB203580, SP600125 and U0126 blocked the effect of LPS on the acetylcholine-induced contractions. Phosphorylated p38 and ERK were detected in neurons of myenteric plexus and Phosphorylated p38 and JNK in smooth muscle cells of ileum.. we can suggest that p38, JNK, and ERK MAPKs are involved in the mechanism of action of LPS in the ileum.

Topics: Acetylcholine; Animals; Anthracenes; Biomarkers; Butadienes; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Ileum; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Male; Mitogen-Activated Protein Kinases; Muscle Contraction; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Rabbits

We investigated the unknown molecular mechanisms of Interleukin-1 (IL-1β)-induced cartilage aggrecan degeneration by aggrecanase (ADAMTS-A Disintegrin And Metalloproteinase with ThromboSpondin motifs) in human articular chondrocytes, a model mimicking human arthritis.. Chondrocytes were pretreated with various pharmacological inhibitors and then stimulated with IL-1β for 24 h. ADAMTS-4 expression or activity was studied by RT-PCR or ELISA and other proteins measured by Western blotting.. MAP kinase kinase-specific inhibitor, U0126 inhibited IL-1-induced phosphorylation of ERK1/2 and down-regulated ADAMTS-4 expression and activity. Protein 38 inhibitor, SB203580 down-regulated the phosphorylation of p38 and its target, activating transcription factor-2 (ATF-2), ADAMTS-4 mRNA and activity. C-Jun N-terminal kinase (JNK) inhibitor, SP600125 diminished IL-1-stimulated JNK phosphorylation, ADAMTS-4 mRNA expression and enzyme activity. A c-fos/lipoxygenase pathway inhibitor and antioxidant, nordihydroguaiaretic acid (NDGA) significantly suppressed ADAMTS-4 mRNA induction and activity. Activating protein (AP-1) and nuclear factor kappa B (NF-ĸB) transcription factor inhibitors, curcumin and pyrrolidine dithiocarbamate (PDTC) partially inhibited ADAMTS-4 induction and activity.. These results suggest partial involvement of ERK-, p38-and JNK-MAPKs as well as AP-1, ATF-2 and NF-ĸB transcription factors in IL-1-induced ADAMTS-4 in chondrocytes. Inhibition of these targets by the specific pharmacological agents could be useful for reducing aggrecanase-driven cartilage resorption in arthritis.

Topics: Anthracenes; Butadienes; Cartilage, Articular; Cells, Cultured; Chondrocytes; Curcumin; Endopeptidases; Gene Expression Regulation; Humans; Imidazoles; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Lipoxygenases; Masoprocol; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Pyrrolidines; RNA, Messenger; Thiocarbamates; Transcription Factor AP-1

We aimed to investigate specific roles of mitogen-activated protein kinases (MAPK) in the deterioration of endothelial function during the progression of diabetes and the potential therapeutic effects of MAPK inhibitors and agonists in the amelioration of endothelial function. Protein expression and phosphorylation of p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular signal-regulated kinase (Erk) were assessed in mesenteric arteries of 3- (3M) and 9-month-old (9M) male diabetic and control mice. The expression of p38, JNK, and Erk was comparable in all groups of mice, but the phosphorylation of p38 and JNK was increased in 3M and further increased in 9M diabetic mice, whereas the phosphorylation of Erk was substantially reduced in 9M diabetic mice. NADPH oxidase-dependent superoxide production was significantly increased in vessels of two ages of diabetic mice. Inhibition of either p38 with SB203580 or JNK with SP600125 reduced superoxide production and improved shear stress-induced dilation (SSID) in 3M, but not in 9M, diabetic mice. Treating the vessels of 9M diabetic mice with resveratrol increased Erk phosphorylation and shear stress-induced endothelial nitric oxide synthase (eNOS) phosphorylation and activity, but resveratrol alone did not improve SSID. Administration of resveratrol and SB203580 or resveratrol and SP600125 together significantly improved SSID in vessels of 9M diabetic mice. The improved response was prevented by U0126, an Erk inhibitor. Thus, p38/JNK-dependent increase in oxidative stress diminished nitric oxide-mediated dilation in vessels of 3M diabetic mice. Oxidative stress and impaired Erk-dependent activation of eNOS exacerbates endothelial dysfunction in the advanced stage of diabetes.

Topics: Animals; Anthracenes; Butadienes; Diabetes Mellitus, Type 2; Extracellular Signal-Regulated MAP Kinases; Imidazoles; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mitogen-Activated Protein Kinases; Nitriles; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Receptors, Leptin; Resveratrol; Signal Transduction; Stilbenes; Superoxides

In this study, we investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in a platelet-activating factor (PAF)-induced experimental pulmonary tumor metastasis model. An adenovirus carrying PTEN cDNA (Ad-PTEN) reversed PAF-induced increase in phosphorylation of AKT as well as pulmonary metastasis of B16F10. PAF-induced pulmonary metastasis was inhibited by MAPK inhibitors, but not by PI3K inhibitor. Ad-PTEN abrogated PAF-induced phosphorylation of MAPKs. These data indicate PTEN/MAPK pathways play a key role in PAF-induced tumor metastasis.

Topics: Androstadienes; Animals; Anthracenes; Butadienes; Cell Line; Humans; Imidazoles; Lung Neoplasms; MAP Kinase Signaling System; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Platelet Activating Factor; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyridines; Wortmannin

The cilium and cilial proteins have emerged as principal mechanosensors of renal epithelial cells responsible for translating mechanical forces into intracellular signals. Polycystin-2 (PC-2), a cilial protein, regulates flow/shear-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) and recently has been implicated in the regulation of mitogen-activated protein (MAP) kinases. We hypothesize that fluid shear stress (FSS) activates PC-2 which regulates MAP kinase and, in turn, induces MAP kinase-dependent gene expression, specifically, monocyte chemoattractant protein-1 (MCP-1).. To test this, PC-2 expression was constitutively reduced in a murine inner medullary collecting duct (IMCD3) cell line, and the expression of FSS-induced MCP-1 expression and MAP kinase signaling compared between the parental (PC-2-expressing) and PC-2-deficient IMCD3 cells.. FSS induces MAP kinase signaling and downstream MCP-1 mRNA expression in wild-type IMCD3 cells, while inhibitors of MAP kinase prevented the FSS-induced MCP-1 mRNA response. In contradistinction, FSS did not induce MCP-1 mRNA expression in PC-2-deficient cells, but did increase activation of the upstream MAP kinases. Wild-type cells exposed to FSS augmented the nuclear abundance of activated MAP kinase while PC-2-deficient cells did not.. PC-2 regulates FSS-induced MAP kinase trafficking into the nucleus of CD cells.

Topics: Animals; Anthracenes; Blotting, Western; Butadienes; Cell Line; Chemokine CCL2; Enzyme Inhibitors; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Fluorescent Antibody Technique; Gene Expression Regulation; JNK Mitogen-Activated Protein Kinases; Kidney Tubules, Collecting; Mice; Mitogen-Activated Protein Kinases; Nitriles; Reverse Transcriptase Polymerase Chain Reaction; Rheology; RNA Interference; Stress, Mechanical; TRPP Cation Channels

α-Tocopherol ether-linked acetic acid (α-TEA) is a promising agent for cancer prevention/therapy based on its antitumour actions in a variety of cancers.. Human breast cancer cells, MCF-7 and HCC-1954, were used to study the effect of α-TEA using Annexin V/PI staining, western blot analyses, and siRNA knockdown techniques.. α-Tocopherol ether-linked acetic acid suppressed constitutively active basal levels of pAKT, pERK, pmTOR, and their downstream targets, as well as induced both cell types to undergo apoptosis. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin suppressed pAKT, pERK, pmTOR, and their downstream targets, indicating PI3K to be a common upstream mediator. In addition, α-TEA induced increased levels of pIRS-1 (Ser-307), a phosphorylation site correlated with insulin receptor substrate-1 (IRS-1) inactivation, and decreased levels of total IRS-1. Small interfering RNA (siRNA) knockdown of JNK blocked the impact of α-TEA on pIRS-1 and total IRS-1 and impeded its ability to downregulate the phosphorylated status of AKT, ERK, and mTOR. Combinations of α-TEA+MEK or mTOR inhibitor acted cooperatively to induce apoptosis and reduce basal levels of pERK and pmTOR. Importantly, inhibition of MEK and mTOR resulted in increased levels of pAKT and IRS-1, and α-TEA blocked them.. Downregulation of IRS-1/PI3K pathways via JNK are critical for α-TEA and α-TEA+MEK or mTOR inhibitor-induced apoptosis in human MCF-7 and HCC-1954 breast cancer cells.

Topics: 1-Phosphatidylinositol 4-Kinase; alpha-Tocopherol; Androstadienes; Anthracenes; Antioxidants; Apoptosis; Blotting, Western; Breast Neoplasms; Butadienes; Drug Synergism; Drug Therapy, Combination; Enzyme Inhibitors; Female; Humans; Immunosuppressive Agents; Insulin Receptor Substrate Proteins; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Wortmannin

Interaction between the immune system and cancer allows for the use of biological response modifiers, e.g. OK-432, in cancer therapy. OK-432, penicillin-killed Streptococcus pyogenes, is used in treating carcinomas, but also lymphangiomas. We have studied the role of monocytes (MOs) in the immune response to OK-432 by examining IL-6 and tumour necrosis factor (TNF)-α secretion after in vitro MO stimulation with OK-432, to some extent in comparison with lipoteichoic acid (LTA) and lipopolysaccharide (LPS). LTA stimulation of whole blood gave IL-6 but not TNF-α secretion, as previously shown with OK-432 stimulation, whereas both cytokines were secreted following LPS stimulation. Addition of the MAPK kinase (MAPKK) MEK inhibitor U0126 inhibited IL-6/TNF-α secretion in a dose-dependent manner. Flow cytometry and to some extent Western blot (Wb) analyses showed that MAPK ERK, located downstream of MEK1/2, is predominantly phosphorylated at isolation from peripheral blood. Addition of the p38 MAP kinase inhibitor SB202190 decreased MO IL-6/TNF-α production upon OK-432 stimulation in a dose-dependent manner. Addition of the MAPK JNK inhibitor SP600125 did not systematically change the MO IL-6/TNF-α OK-432 response. Flow cytometry showed that when stimulating the MOs before isolation from blood, LPS yielded ERK phosphorylation and LPS/LTA p38 phosphorylation, whereas OK-432 had no effects on phosphorylation levels. In conclusion, we have shown that OK-432 resembles TLR2 more than TLR4 stimulation of MOs and depends on MAPKK MEK and MAPK p38, but not on JNK phosphorylation. The MEK and p38 MO OK-432 stimulation dependence is possibly related to the differentiation of cells of the MO lineage.

Topics: Anthracenes; Antineoplastic Agents; Butadienes; Cells, Cultured; Enzyme Inhibitors; Humans; Imidazoles; Interleukin-6; Leukocytes, Mononuclear; Lipopolysaccharides; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Picibanil; Pyridines; Teichoic Acids; Tumor Necrosis Factor-alpha

The angiotensin II type 1 receptor (AT(1)R) is a key regulator of blood pressure and cardiac contractility and is profoundly involved in development of cardiac disease. Since several microRNAs (miRNAs) have been implicated in cardiac disease, we determined whether miRNAs might be regulated by AT(1)R signals in a Gαq/11-dependent or -independent manner.. We performed a global miRNA array analysis of angiotensin II (Ang II)-mediated miRNA regulation in HEK293N cells overexpressing the AT(1)R and focused on separating the role of Gαq/11-dependent and -independent pathways. MiRNA regulation was verified with quantitative PCR in both HEK293N cells and primary cardiac myocytes and fibroblasts.. Our studies revealed five miRNAs (miR-29b, -129-3p, -132, -132* and -212) that were up-regulated by Ang II in HEK293N cells. In contrast, the biased Ang II analogue, [Sar1, Ile4, Ile8] Ang II (SII Ang II), which selectively activates Gαq/11-independent signalling, failed to regulate miRNAs in HEK293N cells. Furthermore, Ang II-induced miRNA regulation was blocked following Gαq/11 and Mek1 inhibition. The observed Ang II regulation of miRNA was confirmed in primary cultures of adult cardiac fibroblasts. Interestingly, Ang II did not regulate miRNA expression in cardiac myocytes, but SII Ang II significantly down-regulated miR-129-3p.. Five miRNAs were regulated by Ang II through mechanisms depending on Gαq/11 and Erk1/2 activation. These miRNAs may be involved in Ang II-mediated cardiac biology and disease, as several of these miRNAs have previously been associated with cardiovascular disease and were found to be regulated in cardiac cells.

Topics: Angiotensin II; Anthracenes; Butadienes; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; GTP-Binding Protein alpha Subunits, Gq-G11; HEK293 Cells; Humans; Imidazoles; MicroRNAs; Myocytes, Cardiac; Nitriles; Pyridines; Receptor, Angiotensin, Type 1; Signal Transduction

The role of mitogen-activated protein kinases (MAPK) in the mechanism of EGF-mediated prevention of acetaldehyde-induced tight junction disruption was evaluated in Caco-2 cell monolayers. Pretreatment of cell monolayers with EGF attenuated acetaldehyde-induced decrease in resistance and increase in inulin permeability and redistribution of occludin, zona occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. EGF rapidly increased the levels of phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK1. Pretreatment of cell monolayers with U-0126 (inhibitor of ERK activation), but not SB-202190 and SP-600125 (p38 MAPK and JNK inhibitors), significantly attenuated EGF-mediated prevention of acetaldehyde-induced changes in resistance, inulin permeability, and redistribution of occludin and ZO-1. U-0126, but not SB-202190 and SP-600125, also attenuated EGF-mediated prevention of acetaldehyde effect on the midregion F-actin ring. However, EGF-mediated preservation of junctional distribution of E-cadherin and β-catenin was unaffected by all three inhibitors. Expression of wild-type or constitutively active MEK1 attenuated acetaldehyde-induced redistribution of occludin and ZO-1, whereas dominant-negative MEK1 prevented EGF-mediated preservation of occludin and ZO-1 in acetaldehyde-treated cells. MEK1 expression did not alter E-cadherin distribution in acetaldehyde-treated cells in the presence or absence of EGF. Furthermore, EGF attenuated acetaldehyde-induced tyrosine-phosphorylation of occludin, ZO-1, claudin-3, and E-cadherin. U-0126, but not SB-202190 and SP-600125, prevented EGF effect on tyrosine-phosphorylation of occludin and ZO-1, but not claudin-3, E-cadherin, or β-catenin. These results indicate that EGF-mediated protection of tight junctions from acetaldehyde requires the activity of ERK1/2, but not p38 MAPK or JNK1/2, and that EGF-mediated protection of adherens junctions is independent of MAPK activities.

Topics: Acetaldehyde; Actins; Adherens Junctions; Anthracenes; beta Catenin; Butadienes; Caco-2 Cells; Cadherins; Claudin-3; Enzyme Inhibitors; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; Inulin; Membrane Proteins; Nitriles; Occludin; Permeability; Phosphoproteins; Pyridines; Tight Junctions; Zonula Occludens-1 Protein

Tissue factor pathway inhibitor 2 (TFPI2) is a serine protease inhibitor critical for the regulation of extracellular matrix remodeling and atherosclerotic plaque stability. Previously, we demonstrated that TFPI2 expression is increased in monocytes from patients with familial combined hyperlipidemia (FCH). To gain insight into the molecular mechanisms responsible for this upregulation, we examined TFPI2 expression in THP-1 macrophages exposed to lipoproteins and thrombin. Our results showed that TFPI2 expression was not affected by treatment with very low density lipoproteins (VLDL), but was induced by thrombin (10 U/ml) in THP-1 (1.9-fold increase, p<0.001) and human monocyte-derived macrophages (2.3-fold increase, p<0.005). The specificity of the inductive effect was demonstrated by preincubation with the thrombin inhibitors hirudin and PPACK, which ablated thrombin effects. TFPI2 induction was prevented by pre-incubation with MEK1/2 and JNK inhibitors, but not by the EGF receptor antagonist AG1478. In the presence of parthenolide, an inhibitor of NFκB, but not of SR-11302, a selective AP-1 inhibitor, thrombin-mediated TFPI2 induction was blunted. Our results also show that thrombin treatment increased ERK1/2, JNK and IκBα phosphorylation. Finally, we ruled out the possibility that TFPI2 induction by thrombin was mediated by COX-2, as preincubation with a selective COX-2 inhibitor did not prevent the inductive effect. In conclusion, thrombin induces TFPI2 expression by a mechanism involving ERK1/2 and JNK phosphorylation, leading finally to NFkB activation. In the context of atherosclerosis, thrombin-induced macrophage TFPI2 expression could represent a means of avoiding excessive activation of matrix metalloproteases at sites of inflammation.

Topics: Amino Acid Chloromethyl Ketones; Anthracenes; Antithrombins; Blotting, Western; Butadienes; Cell Line; Cells, Cultured; Cyclooxygenase 2 Inhibitors; Enzyme Inhibitors; Flavonoids; Gene Expression; Glycoproteins; Hirudins; Humans; JNK Mitogen-Activated Protein Kinases; Lipoproteins, VLDL; Macrophages; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Nitrobenzenes; Reverse Transcriptase Polymerase Chain Reaction; Sesquiterpenes; Signal Transduction; Sulfonamides; Thrombin; Time Factors

N-methyl-D-aspartate (NMDA)-induced pial artery dilation (PAD) is reversed to vasoconstriction after fluid percussion brain injury (FPI). Tissue type plasminogen activator (tPA) is up-regulated and the tPA antagonist, EEIIMD, prevents impaired NMDA PAD after FPI. Mitogen-activated protein kinase (MAPK), a family of at least three kinases, ERK, p38, and JNK, is also up-regulated after traumatic brain injury (TBI). We hypothesize that tPA impairs NMDA-induced cerebrovasodilation after FPI in a MAPK isoform-dependent mechanism.. Lateral FPI was induced in newborn pigs. The closed cranial window technique was used to measure pial artery diameter and to collect cerebrospinal fluid (CSF). ERK, p38, and JNK MAPK concentrations in CSF were quantified by ELISA.. CSF JNK MAPK was increased by FPI, increased further by tPA, but blocked by JNK antagonists SP600125 and D-JNKI1. FPI modestly increased p38 and ERK isoforms of MAPK. NMDA-induced PAD was reversed to vasoconstriction after FPI, whereas dilator responses to papaverine were unchanged. tPA, in post-FPI CSF concentration, potentiated NMDA-induced vasoconstriction while papaverine dilation was unchanged. SP 600125 and D-JNKI1, blocked NMDA-induced vasoconstriction and fully restored PAD. The ERK antagonist U 0126 partially restored NMDA-induced PAD, while the p38 inhibitor SB203580 aggravated NMDA-induced vasoconstriction observed in the presence of tPA after FPI.. These data indicate that tPA contributes to impairment of NMDA-mediated cerebrovasodilation after FPI through JNK, while p38 may be protective. These data suggest that inhibition of the endogenous plasminogen activator system and JNK may improve cerebral hemodynamic outcome post-TBI.

Topics: Animals; Animals, Newborn; Anthracenes; Brain Injuries; Butadienes; Drug Interactions; Female; Imidazoles; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinases; N-Methylaspartate; Nitriles; Oligopeptides; Papaverine; Peptides; Pia Mater; Pyridines; Swine; Tissue Plasminogen Activator; Up-Regulation; Vasoconstriction; Vasodilation

Ovulated eggs possess maternal apoptotic execution machinery that is inhibited for a limited time. The fertilized eggs switch off this time bomb whereas aged unfertilized eggs and parthenogenetically activated eggs fail to stop the timer and die. To investigate the nature of the molecular clock that triggers the egg decision of committing suicide, we introduce here Xenopus eggs as an in vivo system for studying the death of unfertilized eggs. We report that after ovulation, a number of eggs remains in the female body where they die by apoptosis. Similarly, ovulated unfertilized eggs recovered in the external medium die within 72 h. We showed that the death process depends on both cytochrome c release and caspase activation. The apoptotic machinery is turned on during meiotic maturation, before fertilization. The death pathway is independent of ERK but relies on activating Bad phosphorylation through the control of both kinases Cdk1 and JNK. In conclusion, the default fate of an unfertilized Xenopus egg is to die by a mitochondrial dependent apoptosis activated during meiotic maturation.

Topics: Animals; Anthracenes; Apoptosis; bcl-Associated Death Protein; Blotting, Western; Butadienes; Caspases; CDC2 Protein Kinase; Cyclin B; Cytochromes c; Female; JNK Mitogen-Activated Protein Kinases; Mitochondria; Mitogen-Activated Protein Kinase 3; Models, Biological; Molecular Sequence Data; Nitriles; Oocytes; Ovum; Phosphorylation; Progesterone; Time Factors; Xenopus laevis; Xenopus Proteins

Fetal membranes overlying the cervix (i.e. supracervical site, SCS) are characterised by increased extracellular matrix (ECM) degradation. In non-gestational tissues, the mitogen activated protein kinase (MAPK) and activator protein (AP)-1 family are involved in the regulation of the ECM degrading enzyme metalloproteinase (MMP)-9. The aims of this study were (i) to compare the expression of AP-1 proteins in fetal membranes from the SCS and a distal site (DS), and (ii) determine if the MAPK/AP-1 pathway is involved in the regulation of MMP-9. Fetal membranes overlying the cervix were identified in situ in women undergoing term elective Caesarean section. Immunohistochemistry (n = 6) was used to localise the expression of the MAPK proteins ERK (total and phosphorylated), JNK (total and phosphorylated) and p38 MAPK (total and phosphorylated), and the AP-1 proteins JunB, cJun (total and phosphorylated), JunD, cFos and FosD. There was no difference in JNK, p38 MAPK, FosB, cJun and JunD protein expression between SC and distal fetal membranes. However, when compared to DS, the intensity and/or extent of staining of ERK, p-ERK, p-JNK, p-p38 MAPK, cFos, JunB and p-cJun were greater in amnion and chorion obtained from the SCS. In order to elucidate a role for these proteins in ECM degradation, pharmacological inhibitors of MAPK protein activation were utilised in primary amnion cells. The ERK inhibitor U0126, JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190 all significantly decreased IL-β-induced MMP-9 gene expression and pro MMP-9 in human primary amnion cells. In summary, at term, non laboured SC fetal membranes are characterised by increased expression of MAPK and AP-1 proteins. MMP-9 expression and production was significantly suppressed by inhibitors of three key enzymes in the signalling cascades leading to AP-1 formation, ERK 1/2, JNK and p38 MAPK. Thus, the MAPK/AP-1 pathway may play a role in the degradation of the ECM at the SCS making it more susceptible to membrane rupture.

Topics: Anthracenes; Butadienes; Cervix Uteri; Extracellular Signal-Regulated MAP Kinases; Extraembryonic Membranes; Female; Gentian Violet; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinases; Nitriles; p38 Mitogen-Activated Protein Kinases; Pregnancy; Pyridines; Quaternary Ammonium Compounds; Transcription Factor AP-1

Recently, several flavonoids have been shown to have cardioprotective, cancer preventive, or anti-inflammatory properties. However, the specific mechanisms underlying their protective effects remain unclear. We aimed to investigate the different effects of three representative flavonoids-hesperidin, naringin, and resveratrol-on intracellular adhesion molecule-1 (ICAM-1) induction in human umbilical vein endothelial cells (HUVECs) by using high-glucose (HG) concentrations and the possible underlying molecular mechanisms. In HG-induced HUVEC cultures, the effects of three different flavonoids on ICAM-1 production and p38 phosphorylation were examined in the presence or absence of inhibitors targeting the mitogen-activated protein kinase (MAPK) signal transduction pathway. HG stimulation of HUVECs increased the levels of the adhesion molecules ICAM-1 and endothelial selectin (E-selectin). Pretreatment with all the three flavonoids drastically inhibited ICAM-1 expression in a time-dependent manner, but did not alter VCAM-1 and E-selectin expressions. Moreover, we investigated the effects of flavonoids on the MAPK signal transduction pathway, because MAPK families are associated with vascular inflammation under stress. These flavonoids did not block HG-induced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but completely inhibited the HG-induced phosphorylation of p38 MAPK. SB202190, an inhibitor of p38 MAPK, also inhibited the HG-induced enrichment of ICAM-1. This study demonstrated that hesperidin, naringin, and resveratrol reduced the HG-induced ICAM-1 expression via the p38 MAPK signaling pathway, contributing to the inhibition of monocyte adhesion to endothelial cells.

Topics: Anthracenes; Butadienes; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Flavanones; Glucose; Hesperidin; Humans; Hyperglycemia; Imidazoles; Intercellular Adhesion Molecule-1; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Resveratrol; Stilbenes; Up-Regulation; Veins

Cholesterol is one of major components of cell membrane and plays a role in vesicular trafficking and cellular signaling. We investigated the effects of cholesterol on matrix metalloproteinase-2 (MMP-2) activation in human dermal fibroblasts. We found that tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) expression and active form MMP-2 (64 kD) were dose-dependently increased by methyl-beta-cyclodextrin (MbetaCD), a cholesterol depletion agent. In contrast, cholesterol depletion-induced TIMP-2 expression and MMP-2 activation were suppressed by cholesterol repletion. Then we investigated the regulatory mechanism of TIMP-2 expression by cholesterol depletion. We found that the phosphorylation of JNK as well as ERK was significantly increased by cholesterol depletion. Moreover, cholesterol depletion-induced TIMP-2 expression and MMP-2 activation was significantly decreased by MEK inhibitor U0126, and JNK inhibitor SP600125, respectively. While a low dose of recombinant TIMP-2 (100 ng/ml) increased the level of active MMP-2 (64 kD), the high dose of TIMP-2 (>or=200 ng/ml) decreased the level of active MMP-2 (64 kD). Taken together, we suggest that the induction of TIMP-2 by cholesterol depletion leads to the conversion of proMMP-2 (72 kD) into active MMP-2 (64 kD) in human dermal fibroblasts.

Topics: Anthracenes; Butadienes; Cells, Cultured; Child; Child, Preschool; Cholesterol; Cyclodextrins; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Humans; Immunoblotting; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; Matrix Metalloproteinase 2; Microscopy, Electron, Transmission; Nitriles; Tissue Inhibitor of Metalloproteinase-2

Here, we aim to investigate the mechanisms of Toll-like receptor (TLR)-induced prodiabetic and proinflammatory activation of adipocytes and to detect differences in the responsiveness of TLRs to their respective ligands between adipocytes isolated from inflamed vs. noninflamed adipose tissue. Experiments using specific ligands for all known TLRs were performed in murine 3T3-L1 adipocytes and in human adipocytes isolated from noninflamed and inflamed adipose tissue. IL-6 and monocyte chemoattractant protein-1 (MCP-1) release were measured by ELISA. The expression of the signal transduction proteins phospho-extracellular signal-regulated kinase (P-Erk), P-c-Jun N-terminal kinase (JNK), and P-interferon regulatory factor-3 was investigated by Western blot analysis. Additionally, functional inhibitors of MAPK kinase-1/-2 and JNK-1/-2 were used in the stimulation experiments. Activation of TRL4 by lipopolysaccharide (LPS) and TLR1/2 by Pam(3)Cys up-regulates IL-6 and MCP-1 release in adipocytes via specific activation of Erk. Stimulation of adipocytes by macrophage activating lipopeptide-2 (MALP-2) induces MCP-1 but has no effect on IL-6 release. This stimulatory effect on MCP-1 release is antagonized by inhibition of both mitogen-activated protein kinase-1/-2 and JNK-1/-2. Phosphorylation of Erk and JNK is up-regulated after stimulation by MALP-2. In human adipocytes isolated from noninflamed adipose tissue, LPS and Pam(3)Cys, but not MALP-2, are potent inducers of IL-6 and MCP-1. MALP-2 is able to induce IL-6 and MCP-1 release in adipocytes isolated from inflamed adipose tissue, whereas these adipocytes lost their ability to respond to LPS. The present results point to a role of the adipose tissue in innate immunity. TLR-ligand-induced proinflammatory and prodiabetic activation of adipocytes might couple visceral adipose tissue dysfunction with insulin resistance and type 2 diabetes mellitus.

Topics: 3T3-L1 Cells; Adipocytes; Adipose Tissue; Adult; Animals; Anthracenes; Butadienes; Chemokine CCL2; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Inflammation; Insulin; Interferon Regulatory Factor-3; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mice; Myeloid Differentiation Factor 88; Nitriles; Phosphorylation; RNA, Messenger; Toll-Like Receptors

Deferoxamine mesylate is clinically used as a chelating agent but might induce retinopathy. To evaluate its effect on the retinal pigment epithelium (RPE), porcine RPE cells were stimulated with deferoxamine. Cell death was assessed with trypan blue exclusion assay. To investigate the pathway of cell death, the mitogen-activated protein kinases (MAPKs) Erk, JNK, and p38 were inhibited with U0126, SP600125, and SB203580, respectively. Their activity was determined by Western blot. Deferoxamine induces significant cell death in RPE cells, accompanied by phosphorylation of p38 and Erk. Inhibition of p38 attenuates cell death. In conclusion, deferoxamine is directly toxic on RPE cells, its toxicity depending on p38.

Topics: Animals; Anthracenes; Blotting, Western; Butadienes; Cell Death; Cells, Cultured; Deferoxamine; Dose-Response Relationship, Drug; Hydrogen Peroxide; Imidazoles; Iron Chelating Agents; Nitriles; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Pyridines; Retinal Pigment Epithelium; Swine; Trypan Blue

Aberrant overexpression of growth factor receptor-bound protein 7 (GRB7) and its variant GRB7v has been found in numerous human cancers. The goal of this study was to characterize the functions of GRB7 and GRB7v in the ovarian carcinogenesis and to investigate the differential roles of GRB7 and GRB7v in the modulation of signaling pathways.. Quantitative reverse transcription-PCR, Western blot, and immunohistochemical analyses were used to evaluate the levels of GRB7 and GRB7v. The cellular localization, functions, and signaling pathways regulated by GRB7 and GRB7v were investigated by enforced expression of GRB7 and GRB7v.. Quantitative reverse transcription-PCR and Western blot analyses showed that GRB7 and GRB7v were frequently upregulated in ovarian cancer samples. The overexpressed GRB7 (P = 0.009) and GRB7v (P = 0.017) were significantly correlated with high-grade ovarian cancer. Immunohistochemical analysis on ovarian cancer tissue array confirmed that the upregulated GRB7 was significantly correlated with high-grade ovarian cancer (P = 0.001). Confocal microscopy analysis showed that GRB7 and GRB7v predominately localized in cytoplasm of ovarian cancer cells, consistent with their roles as signaling adaptors. Enforced expression of GRB7 promoted cell proliferation, migration, and invasion, whereas GRB7v only increased cell proliferation and anchorage-independent growth ability. With the treatment of specific kinase inhibitors, we showed that both GRB7 and GRB7v promoted cell proliferation through activating extracellular signal-regulated kinase signaling, whereas GRB7 enhanced cell migration/invasion by activating c-Jun NH(2) terminal kinase signaling.. Our studies implicate that the overexpressed GRB7 and GRB7v are associated with high-grade tumors and exert distinct tumorigenic functions through regulating different signaling pathways in ovarian cancer cells.

Topics: Alternative Splicing; Anthracenes; Blotting, Western; Butadienes; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Neoplastic; GRB7 Adaptor Protein; Green Fluorescent Proteins; Humans; Immunohistochemistry; Microscopy, Confocal; Middle Aged; Mitogen-Activated Protein Kinases; Neoplasm Staging; Nitriles; Ovarian Neoplasms; Protein Isoforms; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection

Renal (pro)renin receptor (PRR) expression is increased in diabetes. The exact mechanisms involved in this process are not well established. We hypothesized that high glucose up-regulates PRR through protein kinase C (PKC)-Raf-ERK and PKC-c-Jun N-terminal kinase (JNK)-c-Jun signaling pathways. Rat mesangial cells exposed to 30 mm d-glucose demonstrated significant increase in PRR mRNA and protein expression, intracellular phosphorylation of Raf-1 (Y340/341), ERK, JNK, nuclear factor-kappaB (NF-kappaB) p65 (S536) and c-Jun (S63). By chromatin immunoprecipitation assay and EMSA, high glucose induced more functional NF-kappaB and activator protein (AP)-1 dimers bound to corresponding cis-regulatory elements in the predicted PRR promoter to up-regulate PRR transcription. Conventional and novel PKC inhibitors Chelerythrine and Rottlerin, Raf-1 inhibitor GW5074, MEK1/2 inhibitor U0126, JNK inhibitor SP600125, NF-kappaB inhibitor Quinazoline, and AP-1 inhibitor Curcumin, respectively, attenuated glucose-induced PRR up-regulation. Chelerythrine and Rottlerin also inhibited glucose-induced phosphorylation of Raf-1 (Y340/341), ERK1/2, JNK, NF-kappaB p65 (S536), and c-Jun (S63). GW5074 and U0126 inhibited the phosphorylation of ERK1/2 and NF-kappaB p65 (S536). SP600125 inhibited phosphorylation of NF-kappaB p65 (S536) and c-Jun (S63). We conclude that high glucose up-regulates the expression of PRR through mechanisms dependent on both PKC-Raf-ERK and PKC-JNK-c-Jun signaling pathways. NF-kappaB and AP-1 are involved in high-glucose-induced PRR up-regulation in rat mesangial cells.

Topics: Animals; Anthracenes; Butadienes; Cell Line; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Glucose; Indoles; MAP Kinase Kinase Kinases; Mesangial Cells; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Phenols; Phosphorylation; Promoter Regions, Genetic; Prorenin Receptor; Protein Binding; Proto-Oncogene Proteins c-raf; Rats; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription Factor AP-1

Vascular endothelial growth factor (VEGF) stimulated fetoplacental artery endothelial (oFPAE) cell migration and activated multiple signaling pathways including ERK2/1, p38MAPK, Jun N-terminal kinase (JNK1/2), v-Akt murine thymoma viral oncogene homolog 1 (Akt1), and c-Src in oFPAE cells. VEGF-induced cell migration was blocked by specific kinase inhibitors of JNK1/2 (SP600125), c-Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and phosphatidylinositol 3-kinase/Akt (wortmannin) but not ERK2/1 (U0126) and p38MAPK (SB203580). VEGF-induced cell migration was associated with dynamic actin reorganization and focal adhesion as evidenced by increased stress fiber formation and phosphorylation of cofilin-1 and focal adhesion kinase (FAK) and paxillin. Inhibition of JNK1/2, c-Src, and phosphatidylinositol 3-kinase/Akt suppressed VEGF-induced stress fiber formation and cofilin-1 phosphorylation. c-Src inhibition suppressed VEGF-induced phosphorylation of focal adhesion kinase, paxillin, and focal adhesion. VEGF-induced cell migration requires endogenous nitric oxide (NO) as: 1) VEGF-stimulated phosphorylation of endothelial NO synthase (eNOS) via activation of Akt, JNK1/2, and Src; 2) a NO donor diethylenetriamine-NO-stimulated cell migration; and 3) NO synthase inhibition blocked VEGF-induced cell migration. Targeted down-regulation and overexpression of caveolin-1 both inhibited VEGF-induced cell migration. Caveolin-1 down-regulation suppressed VEGF-stimulated phosphorylation of Akt, JNK, eNOS, c-Src, and FAK; however, basal activities of c-Src and FAK were elevated in parallel with increased stress fiber formation and focal adhesion. Caveolin-1 overexpression also inhibited VEGF-induced phosphorylation of Akt, JNK, c-Src, FAK, and eNOS. Thus, VEGF-induced placental endothelial cell migration requires activation of complex pathways that are paradoxically regulated by caveolin-1.

Topics: Androstadienes; Animals; Anthracenes; Arteries; Butadienes; Caveolin 1; Cell Movement; Cells, Cultured; CSK Tyrosine-Protein Kinase; Electrophoresis, Polyacrylamide Gel; Endothelial Cells; Enzyme Inhibitors; Female; Focal Adhesion Protein-Tyrosine Kinases; Imidazoles; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitric Oxide Synthase Type III; Nitriles; Nitrogen Oxides; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Placenta; Pregnancy; Protein-Tyrosine Kinases; Pyridines; Pyrimidines; Sheep; Signal Transduction; src-Family Kinases; Vascular Endothelial Growth Factor A; Wortmannin

Oxidized low density lipoprotein (oxLDL) is involved in the development of vascular diseases. Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glyero-3-phosphorylcholine) is a key component of oxLDL.. In the present study, we evaluate whether oxLDL can regulate migration of human bone-marrow derived stem cells (hBMSCs) and characterize the role of PAF in the oxLDL-induced migration.. oxLDL stimulated chemotaxis of hBMSCs in vitro. Treatment of the cells with BN52021, a specific antagonist of PAF receptor (PAF-R), completely blocked the cell migration induced by PAF, but not platelet-derived growth factor (PDGF-BB). Using PAF-R-specific small interfering RNA, it was demonstrated that silencing of endogenous PAF-R expression significantly attenuated cell migration induced by PAF, but not PDGF-BB, suggesting the specific involvement of PAF-R in the oxLDL-induced cell migration. In addition, PAF-induced migration of hBMSCs was abrogated by pretreating cells with mitogen-activated protein kinase (MAPK) inhibitors, including the MEK inhibitor U0126, the p38 MAPK inhibitor SB202190, and the JNK inhibitor SP600125. Moreover, adenoviral overexpression of a dominant negative mutant of p38 MAPK blocked PAF-stimulated migration. Taken together, these results suggest that PAF plays a pivotal role in the oxLDL-induced recruitment of hBMSCs through mechanisms involving PAF-R-dependent activation of MAPKs.

Topics: Anthracenes; Becaplermin; Bone Marrow Cells; Butadienes; Cell Movement; Humans; Imidazoles; Lipoproteins, LDL; Mesenchymal Stem Cells; Mitogen-Activated Protein Kinases; Nitriles; p38 Mitogen-Activated Protein Kinases; Platelet Activating Factor; Platelet Membrane Glycoproteins; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-sis; Pyridines; Receptors, G-Protein-Coupled; RNA Interference; RNA, Small Interfering

To investigate the effects of simvastatin on lipopolysaccharides (LPS) induced upregulation of Lp-PLA(2) in human peripheral blood monocytes-macrophages and the related mechanisms.. Peripheral blood monocytes of healthy volunteer were isolated and incubated for 2-3 days. Monocytes were incubated with various concentrations of LPS for 6 h or with 1 µg/ml of LPS for different times in LPS group. In simvastatin group and MAPK inhibitors groups, cells were pre-treated with simvastatin (10(-2) - 10(-7) mmol/L) or various MAPK inhibitors (10 µmol/L SB203580, 20 µmol/L U0126, and 20 µmol/L SP600125) before LPS co-incubation. Lp-PLA(2) activity was measured by chronometry, Lp-PLA(2) mRNA expression was detected by RT-PCR. Protein expressions of Lp-PLA(2) and p38MAPK and phosphorylated p38MAPK were examined by Western blot.. (1) LPS significantly upregulated Lp-PLA(2) mRNA and protein expression, as well as the enzyme activity in a time and concentration dependent manner, which could be significantly attenuated by simvastatin in a time and concentration dependent manner. (2) Simvastatin significantly reduced LPS-induced p38MAPK phosphorylation. The p38 MAPK inhibitor SB203580, but not MEK1/2 inhibitor U0126 and JNK inhibitor SP600125, completely prevented LPS-mediated up-regulation of Lp-PLA(2) at protein level.. This study demonstrated that LPS significantly upregulated Lp-PLA(2) mRNA and protein expression, as well as the enzyme activity in a time and concentration dependent manner via Rho-p38MAPK pathway, which could be significantly suppressed by simvastatin.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Anthracenes; Butadienes; Cells, Cultured; Humans; Imidazoles; Lipopolysaccharides; Macrophages; Monocytes; Nitriles; p38 Mitogen-Activated Protein Kinases; Phospholipases A2; Phosphorylation; Pyridines; RNA, Messenger; Simvastatin

Pancreatic fibrosis is one of the clinical manifestations of chronic pancreatitis and pancreatic cancer. Pancreatic stellate cells (PSCs) have been recognised as principal effector cells in the development of pancreatic fibrosis. The ability to specifically address PSCs might offer a potential for developing a targeted therapy for pancreatic fibrosis.. Characterisation of the 2.2kb hGFAP (human glial fibrillary acidic protein) promoter for its usefulness to express reporter genes specifically in PSCs in vitro and in vivo.. 2.2kb hGFAP-LacZ reporter expressions were examined in four immortalised PSC lines and two non-PSCs, meanwhile, GFAP-LacZ transgenic mice were used to detect LacZ reporter in pancreas tissue. Several kinase inhibitors, vitamin A and its metabolites were applied to study the regulation of 2.2kb hGFAP promoter in PSCs.. Our results showed that the 2.2kb hGFAP promoter is capable of regulating the expression of reporter genes exclusively in immortalised and primary PSCs, as well as in PSCs of transgenic GFAP-LacZ mice. When a PSC cell line transfected with the LacZ reporter (SAM-K/LacZ/C1) was treated with different anti-fibrotic agents and kinase inhibitors, the transgenic beta-galactosidase activity was found to be regulated by multiple signalling pathways known to be involved in the PSC activation.. This study provides the proof of concept for using the 2.2kb hGFAP promoter to specifically manipulate PSCs for the development of targeted gene and/or drug therapy in pancreatic fibrosis, and for the screening of anti-fibrotic agents.

Topics: Animals; Anthracenes; Butadienes; Cell Line; Chromones; Enzyme Inhibitors; Gene Expression; Glial Fibrillary Acidic Protein; Lac Operon; Mice; Mice, Transgenic; Morpholines; Nitriles; Pancreas; Promoter Regions, Genetic; Transfection; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Vitamin A; Vitamins

Accumulating evidence indicates a pivotal role for neuroinflammation in ischemic and excitotoxic brain injury. Cytokine-induced neutrophil chemoattractant-1 (CINC-1) is a CXC chemokine implicated in the infiltration of inflammatory cells into the brain parenchyma. In this study, we investigated the effect of N-methyl-D-aspartate (NMDA)-induced neuronal injury on CINC-1 production in the organotypic cortico-striatal slice cultures. Treatment with 50 microM NMDA for 3 - 4 h caused devastating neuronal damage and increased CINC-1 production. Immunohistochemical analysis revealed that the CINC-1 immunoreactivity was predominantly detected in astrocytes. NMDA failed to induce CINC-1 production in enriched astrocyte cultures or neuron-depleted slice cultures, suggesting that NMDA acted on neuronal cells to induce astrocytic CINC-1 production. NMDA-induced CINC-1 mRNA expression was significantly inhibited by U0126, a mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. These results suggest that NMDA-evoked neuronal injury induced astrocytic CINC-1 production via a MEK/ERK signaling pathway. Manipulation of this signaling pathway may serve as a target for suppressing neuroinflammation and, thereby, treating ischemic brain injury.

Topics: Animals; Anthracenes; Astrocytes; Butadienes; Cells, Cultured; Cerebral Cortex; Chemokine CXCL1; Corpus Striatum; Dizocilpine Maleate; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Imidazoles; Immunochemistry; Mitogen-Activated Protein Kinases; N-Methylaspartate; Neurons; Nitriles; Pyridines; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

Pericyte loss is a cardinal feature of early diabetic retinopathy. We previously reported that highly oxidized-glycated low density lipoprotein (HOG-LDL) induces pericyte apoptosis in vitro. In this study, we investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathways in HOG-LDL-induced apoptosis in human pericytes.. Human retinal capillary pericytes (HRCP) were exposed to native LDL (N-LDL) and HOG-LDL, and apoptosis was measured using flow cytometry. Time- and dose-dependent responses of extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) following exposure to N-LDL or HOG-LDL were determined using western blotting. U0126 (ERK inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) were used to determine the role of MAPK signaling in HOG-LDL-induced apoptosis.. HOG-LDL induced apoptosis in HRCP in a dose-dependent manner at concentrations from 5 to 50 mg/l, with a constant effect from 50 to 200 mg/l. When compared to serum-free medium (SFM), this effect of HOG-LDL was found to be significant at all doses above 10 mg/l. In contrast, N-LDL at 200 mg/l did not induce apoptosis compared with SFM. Exposure to N-LDL versus HOG-LDL induced similar phosphorylation of ERK, p38, and JNK, peaking at 5 min, with similar dose-dependent responses up to 25 mg/l that were constant from 25 to 100 mg/l. Blocking of the ERK, p38, and JNK pathways did not inhibit pericyte apoptosis induced by HOG-LDL.. Our data suggest that apoptosis induced by HOG-LDL in HRCP is independent of the activation of MAPK signaling pathways.

Topics: Anthracenes; Apoptosis; Butadienes; Capillaries; Extracellular Signal-Regulated MAP Kinases; Glycation End Products, Advanced; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lipoproteins, LDL; MAP Kinase Signaling System; Nitriles; p38 Mitogen-Activated Protein Kinases; Pericytes; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Retinal Vessels; Time Factors

The decidual microenvironment is characterized by a unique population of leukocytes composed primarily of CD56(bright) NK cells and macrophages. The latter are situated near trophoblast cells at the fetal-maternal interface and there is evidence that trophoblast cells are capable of recruiting macrophages to this site. This study sought to determine the role of tumour necrosis factor alpha (TNF) in the trophoblast-mediated recruitment of monocyte-derived macrophages to the fetal-maternal interface. The human first trimester extravillous trophoblast cell line HTR-8/SVneo was shown to express TNFR1 and to secrete the monocyte-attracting chemokines CCL2 and CCL5 after exposure to TNF in a dose-dependent manner. TNF-mediated stimulation of CCL2 secretion was completely inhibited by incubating the trophoblast cells with the p38-MAPK inhibitor SB203580, whereas CCL5 secretion was inhibited by treating the trophoblast cells with inhibitors specific for JNK (SP600125) and ERK kinase (U0126). Media conditioned by TNF-treated trophoblast cells significantly enhanced the ability of the monocyte cell line THP-1 to invade through Matrigel, and this effect was inhibited using antibodies specific for CCL2 and CCL5. These results support a role for TNF at the fetal-maternal interface as a regulator of macrophage recruitment by trophoblast cells.

Topics: Anthracenes; Butadienes; Cell Line; Cell Movement; Chemokine CCL2; Chemokine CCL5; Collagen; Culture Media, Conditioned; Drug Combinations; Enzyme Inhibitors; Female; Humans; Imidazoles; In Vitro Techniques; Laminin; Macrophages; MAP Kinase Signaling System; Monocytes; Nitriles; Pregnancy; Proteoglycans; Pyridines; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Trophoblasts; Tumor Necrosis Factor-alpha

Because macrophages play a major role in atherosclerotic plaque destabilization, selective removal of macrophages represents a promising approach to stabilize plaques. We showed recently that the protein synthesis inhibitor cycloheximide, in contrast to puromycin, selectively depleted macrophages in rabbit atherosclerotic plaques without affecting smooth muscle cells (SMCs). The mechanism of action of these two translation inhibitors is dissimilar and could account for the differential effects on SMC viability. It is not known whether selective depletion of macrophages is confined to cycloheximide or whether it can also be achieved with translation inhibitors that have a similar mechanism of action. Therefore, in the present study, we investigated the effect of anisomycin, a translation inhibitor with a mechanism of action similar to cycloheximide, on macrophage and SMC viability. In vitro, anisomycin induced apoptosis of macrophages in a concentration-dependent manner, whereas SMCs were only affected at higher concentrations. In vivo, anisomycin selectively decreased the macrophage content of rabbit atherosclerotic plaques through apoptosis. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] prevented anisomycin-induced macrophage death, without affecting SMC viability. SB202190 decreased anisomycin-induced p38 MAPK phosphorylation, did not alter c-Jun NH(2)-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. The latter effect was abolished by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene ethanolate], although the prevention of anisomycin-induced macrophage death by SB202190 remained unchanged. The JNK phosphorylation inhibitor SP600125 did not affect anisomycin-induced macrophage or SMC death. In conclusion, anisomycin selectively decreased the macrophage content in rabbit atherosclerotic plaques, indicating that this effect is not confined to cycloheximide. p38 MAPK, but not ERK1/2 or JNK, plays a major role in anisomycin-induced macrophage death.

Topics: Animals; Anisomycin; Anthracenes; Aorta; Apoptosis; Butadienes; Carotid Arteries; Carotid Stenosis; Cell Line, Tumor; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Imidazoles; Macrophages; Macrophages, Alveolar; Mice; Mitogen-Activated Protein Kinases; Myocytes, Smooth Muscle; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Rabbits; Tunica Intima; Tunica Media

Polychlorinated biphenyls (PCBs) are reported to induce the formation of reactive oxygen species (ROS) in human neutrophil granulocytes through the activation of the NADPH oxidase. The purpose of the present study is to elucidate the cellular mechanisms responsible for the activation of the NADPH oxidase after exposure to PCB. We have previously shown that PCB activates human neutrophil granulocytes through a calcium dependent activation of phospholipase D and/or phospholipase C, followed by the activation of protein kinase C. In the present study, pharmacological characterization of Aroclor (A) 1242-induced respiratory burst in human neutrophils was conducted by the use of enzymatic inhibitors. Pre-incubation with U0126, SB203580, SP600125, cyclosporin A and FK506 attenuated the A 1242-induced respiratory burst, measured by DCF-fluorescence, and luminol-amplified chemiluminescence. Our results show that the Erk1/2 kinases and p38MAPK/JNK are involved in ROS formation in neutrophils exposed to A 1242.

Topics: Anthracenes; Aroclors; Butadienes; Cyclosporine; Environmental Pollutants; Enzyme Activation; Enzyme Inhibitors; Humans; Imidazoles; Luminescent Measurements; NADPH Oxidases; Neutrophil Activation; Neutrophils; Nitriles; Pyridines; Respiratory Burst; Tacrolimus

The cytotoxic effects of fluoride on odontoblasts are not clear. In this study, we examined whether NaF induces apoptosis in MDPC-23 odontoblast-like cells and the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in NaF-induced apoptosis. MDPC-23 cells incubated with 5 mM NaF for 24 hrs exhibited caspase-3 activation, cleavage of poly(ADP-ribose) polymerase, DNA fragmentation, and an increase in cytoplasmic nucleosomes. Prior to the induction of apoptosis, all MAPKs examined were phosphorylated, but in a different manner. In contrast to the sustained phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38, NaF exposure induced a biphasic phosphorylation of extracellular signal-regulated protein kinase (ERK). NaF-induced apoptosis was markedly suppressed by treatment with the JNK inhibitor, SP600125, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Inhibition of p38 activity did not protect cells from apoptosis. Thus, exposure to NaF induces apoptosis in odontoblast-like cells, depending on JNK and, less significantly, ERK pathways.

Topics: Animals; Anthracenes; Apoptosis; Butadienes; Caspase 3; Cell Culture Techniques; Cell Line; Cell Survival; DNA Fragmentation; Enzyme Activators; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Nitriles; Nucleosomes; Odontoblasts; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Sodium Fluoride; Time Factors

Vascular endothelial growth factor (VEGF) is a fundamental factor for angiogenesis. It plays important roles in pathological conditions (e.g. the development of wet AMD), but also in the healthy organism) e.g. in maintaining the vasculature and supporting the retina). Recent therapies to treat the wet AMD focus on neutralizing VEGF indiscriminately. VEGF is constitutively expressed in the retina, but its expression is upregulated by various (noxious) stimuli, e.g. oxidative stress or hypoxia. Discrimination between constitutive expression of VEGF and its pathological upregulation might provide the possibility of focusing on inhibiting the pathological expression only. Here, we focused on the influence of different mitogen-activated protein kinase (MAPK) (p38, Erk, JNK) on the secretion and expression of VEGF, with or without being challenged by oxidative stress.. VEGF secretion was measured using a perfusion organ culture model; expression was examined in primary RPE culture and Western blotting.. Constitutive VEGF expression and secretion can be diminished by inhibiting p38, while inhibiting Erk or JNK does not show a significant effect. When challenged with oxidative stress (250 microM t-butylhydroperoxide), VEGF expression and secretion increases and the influence of the MAPK changes: While p38 still accounts for about 30% of the secretion, Erk shows a similar influence. Inhibiting JNK presents conflicting results. In organ culture, inhibiting JNK significantly increases VEGF secretion after stimulation with 250 microM tBH, while with regard to VEGF expression in RPE cell culture, this effect could not be seen.. Constitutive and oxidative stress induced VEGF secretion, and expression is differently regulated, which might offer an opportunity to selectively inhibit pathological VEGF expression only.

Topics: Animals; Anthracenes; Blotting, Western; Butadienes; Cell Culture Techniques; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Organ Culture Techniques; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Retinal Pigment Epithelium; Swine; tert-Butylhydroperoxide; Vascular Endothelial Growth Factor A

Tight junctions (TJs) form a barrier to the paracellular diffusion of ions and solutes across epithelia. Although transmembrane proteins of the claudin family have emerged as critical determinants of TJ permeability, little is known about the signaling pathways that control their expression. The aim of this study was to assess the role of three mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH(2)-terminal kinases (JNKs), and p38 kinases, in the regulation of epithelial barrier function and claudin expression in mammary epithelial cells. Addition of either PD169316 (a p38 inhibitor) or SP600125 (a JNK inhibitor) induced formation of domes (a phenomenon dependent on TJ barrier function) and enhanced transepithelial electrical resistance, whereas U0126 (an inhibitor of the ERK1/2 activators MEK1/MEK2) had no significant effect. Similar results were obtained using mechanistically unrelated p38 or JNK inhibitors. PD169316 increased the expression of claudin-4 and -8, whereas SP600125 increased claudin-4 and -9 and downregulated claudin-8. Silencing of p38alpha by isoform-specific small interfering RNAs increased claudin-4 and -8 mRNAs, whereas silencing of p38beta only increased claudin-4 mRNA. Silencing of either JNK1 or JNK2 increased claudin-9 mRNA expression while decreasing claudin-8 mRNA. Moreover, selective silencing of JNK2 increased claudin-4 and -7 mRNAs. Finally, both PD169316 and SP600125 inhibited the paracellular diffusion of Na(+) and Cl(-) across epithelial monolayers. Collectively, these results provide evidence that inhibition of either p38 or JNK enhances epithelial barrier function by selectively modulating claudin expression, implying that the basal activity of these MAPKs exerts a tonic effect on TJ ionic permeability.

Topics: Animals; Anthracenes; Butadienes; Cell Line; Dogs; Epithelium; Female; Imidazoles; Mammary Glands, Animal; MAP Kinase Kinase 4; Membrane Proteins; Mice; Nitriles; p38 Mitogen-Activated Protein Kinases

Inorganic phosphate (Pi) transport probably represents an important function of bone-forming cells in relation to extracellular matrix mineralization. In the present study, we investigated the effect of prostaglandin D2 (PGD2) on Pi transport activity and its intracellular signaling mechanism in MC3T3-E1 osteoblast-like cells. PGD2 stimulated Na-dependent Pi uptake time- and dose-dependently in MC3T3-E1 cells during their proliferative phase. A protein kinase C (PKC) inhibitor calphostin C partially suppressed the stimulatory effect of PGD2 on Pi uptake. The selective inhibitors of mitogen-activated protein (MAP) kinase pathways such as ERK, p38 and Jun kinases suppressed PGD2-induced Pi uptake. The inhibitors of phosphatidylinositol (PI) 3-kinase and S6 kinase reduced this effect of PGD2, while Akt kinase inhibitor did not. These results suggest that PGD2 stimulates Na-dependent Pi transport activity in the phase of proliferation of osteoblasts. The mechanisms responsible for this effect are activation of PKC, MAP kinases, PI 3-kinase and S6 kinase.

Topics: 3T3 Cells; Animals; Anthracenes; Butadienes; Cells, Cultured; Chromones; Dose-Response Relationship, Drug; Imidazoles; Mice; Morpholines; Naphthalenes; Nitriles; Osteoblasts; Phosphates; Prostaglandin D2; Pyridines; Signal Transduction; Sirolimus; Sodium

The kinase TAK1, a mitogen-activated protein kinase kinase kinase (MAP3K), has been widely accepted as a key kinase activating NF-kappaB and MAPKs in tumor necrosis factor alpha (TNF-alpha) signaling. We have recently reported that TAK1 regulates the transient phosphorylation and endocytosis of epidermal growth factor receptor (EGFR) in a tyrosine kinase activity-independent manner. In the present study, we found that Thr-669 in the juxtamembrane domain and Ser-1046/1047 in the carboxyl-terminal regulatory domain were transiently phosphorylated in response to TNF-alpha. Experiments using chemical inhibitors and small interfering RNA demonstrated that TNF-alpha-mediated phosphorylation of Thr-669 and Ser-1046/7 were differently regulated via TAK1-extracellular signal-regulated kinase (ERK) and TAK1-p38 pathways, respectively. In addition, p38, but not ERK, was involved in the endocytosis of EGFR. Surprisingly, modified EGFR was essential to prevent apoptotic cellular responses; however, the EGFR pathway was independent of the NF-kappaB antiapoptotic pathway. These results demonstrated that TAK1 controls two different signaling pathways, IkappaB kinase-NF-kappaB and MAPK-EGFR, leading to the survival of cells exposed to the death signal from the TNF-alpha receptor.

Topics: Anthracenes; Butadienes; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Imidazoles; Lactones; MAP Kinase Kinase Kinases; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Quinazolines; Resorcinols; RNA, Small Interfering; Serine; Signal Transduction; Threonine; Tumor Necrosis Factor-alpha

Enterobacter sakazakii (ES) is an emerging pathogen that causes meningitis and necrotizing enterocolitis in infants. Dendritic cells (DCs) are professional phagocytic cells that play an essential role in host defense against invading pathogens; however, the interaction of ES with DCs is not known. In this study, we demonstrate that ES targets DC-specific ICAM nonintegrin (DC-SIGN) to survive in myeloid DCs for which outer membrane protein A (OmpA) expression in ES is critical, although it is not required for uptake. In addition, DC-SIGN expression was sufficient to cause a significant invasion by ES in HeLa cells and intestinal epithelial cells, which are normally not invaded by ES. OmpA(+) ES prevented the maturation of DCs by triggering the production of high levels of IL-10 and TGF-beta and by suppressing the activation of MAPKs. Pretreatment of DCs with Abs to IL-10 and TGF-beta or of bacteria with anti-OmpA Abs significantly enhanced the maturation markers on DCs. Furthermore, DCs pretreated with various inhibitors of MAPKs prohibited the increased production of proinflammatory cytokines stimulated by LPS or OmpA(-) ES. LPS pretreatment followed by OmpA(+) ES infection of DCs failed to induce maturation of DCs, indicating that OmpA(+) ES renders the cells in immunosuppressive state to external stimuli. Similarly, OmpA(+) ES-infected DCs failed to present Ag to T cells as indicated by the inability of T cells to proliferate in MLR. We conclude that ES interacts with DC-SIGN to subvert the host immune responses by disarming MAPK pathway in DCs.

Topics: Animals; Anthracenes; Bacterial Outer Membrane Proteins; Butadienes; Cell Adhesion Molecules; Cell Survival; Cronobacter sakazakii; Dendritic Cells; Enterobacteriaceae Infections; Enzyme Inhibitors; Epithelial Cells; Flavonoids; HeLa Cells; Humans; Imidazoles; Interleukin-10; Lectins, C-Type; Lipopolysaccharides; Lymphocyte Activation; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Mitogen-Activated Protein Kinase Kinases; Nitriles; Pyridines; Rats; Receptors, Cell Surface; Transfection; Transforming Growth Factor beta

This study evaluated the proliferation effects of huangqi on neuron regeneration. We investigated the molecular mechanisms, which include: (1) cyclin D1, A, E-cell cycle factors and MAPK signaling proliferation (2) FGF-2-UPA-MMPs migration signaling. After treatment with various Huanqi concentrations (1.25, 12.5, 125, 250 and 500 microg/ml,), we observed that Huanqi can increase Rsc 96 cell proliferation at 12.5 microg/ml (p < 0.01) concentration determined by the MTT and wound healing tests. Examination by RT-PCR and Western blotting assay showed that Huangqi is able to stimulate the mRNA and protein expressions of cyclin D1, A, E, cell cycle controlling proteins and excite ERK and P38 MAPK signaling pathways to promote cell proliferation. Huangqi stimulates the FGF-2-UPA-MMP 9 migration pathway and enhances RSC 96 Schwann cells migration. Using MAPK chemical inhibitors, U0126, SB203580 and SP600125, the proliferative effects of Huangqi on RSC 96 cells were ERK and P38 signaling-dependent. Based on these results, applying an appropriate dose of Huangqi with biomedical materials would be a potential approach to enhancing neuron regeneration.

Topics: Animals; Anthracenes; Astragalus Plant; Astragalus propinquus; Blotting, Western; Butadienes; Cell Line; Cell Movement; Cell Proliferation; Cell Survival; Cyclin A; Cyclin D1; Cyclin E; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Enzyme Inhibitors; Fibroblast Growth Factor 2; Imidazoles; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase 3; Models, Biological; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Reverse Transcriptase Polymerase Chain Reaction; Schwann Cells; Urokinase-Type Plasminogen Activator

Mitogen-activated protein kinases (MAPKs) are important regulators of aryl hydrocarbon receptor (AhR). An immense progress in MAPKs' biochemistry was attained with the discovery of their specific pharmacological inhibitors. Unfortunately, the inhibitors of JNK and ERK MAPKs, i.e. SP600125 and U0126, respectively, affect AhR-CYP1A signaling pathway because they are partial agonists of AhR and induce CYP1A genes. This implies that SP600125 and U0126 are inappropriate tools for studies of the role of MAPKs in AhR regulation. The results from studies using SP600125 or U126, past or future, should be interpreted with prudence regarding their stimulatory effects on AhR-CYP1A pathway.

Topics: Anthracenes; Butadienes; Cells, Cultured; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Extracellular Signal-Regulated MAP Kinases; Hepatocytes; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Nitriles; p38 Mitogen-Activated Protein Kinases; Pharmaceutical Preparations; Pyridines; Receptors, Aryl Hydrocarbon

EphBs receptors and ephrinBs ligands are present in the adult brain and peripheral tissue and play a critical role in modulating multiple aspects of physiology and pathophysiology. Ours and other studies have demonstrated that spinal ephrinBs/EphBs signaling was involved in the modulation of nociceptive information and central sensitization. However, the role of ephrinBs/EphBs signaling in peripheral sensitization is poorly understood. This study shows that intraplantar (i.pl.) injection of ephrinB1-Fc produces a dose- and time-dependent thermal and mechanical hyperalgesia and the increase of spinal Fos protein expression in mice, which can be partially prevented by pre-treatment with EphB1-Fc. EphrinB1-Fc-induced hyperalgesia is accompanied with the NMDA receptor-mediated increase of expression in peripheral and spinal phosphorylated mitogen-activated protein kinases (phospho-MAPKs) including p-p38, pERK and pJNK, and also is prevented or reversed by the inhibition of peripheral and spinal MAPKs. Furthermore, in formalin inflammation pain model, pre-inhibition of EphBs receptors by the injection of EphB1-Fc reduces pain behavior, which is accompanied by the decreased expression of peripheral p-p38, pERK and pJNK. These data provide evidence that ephrinBs may act as a prominent contributor to peripheral sensitization, and demonstrate that activation of peripheral ephrinBs/EphBs system induces hyperalgesia through a MAPKs-mediated mechanism.

Topics: Animals; Anthracenes; Butadienes; Dizocilpine Maleate; Ephrin-B1; Foot; Formaldehyde; Hot Temperature; Hyperalgesia; Injections, Spinal; Male; MAP Kinase Signaling System; Mice; Nitriles; p38 Mitogen-Activated Protein Kinases; Physical Stimulation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-fos; Receptors, Eph Family; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Touch

Glutaric (GA) and 3-hydroxyglutaric (OHGA) acids accumulate in glutaric acidemia I (GAI), a neurometabolic disease characterized by acute striatal degeneration and chronic progressive cortical atrophy. To explore the hypothesis that astrocytes are involved in GAI pathogenesis and targets of accumulating metabolites, we determined the effects of GA and OHGA on cultured rat cortical astrocytes. Remarkably, both acids induced mitochondria depolarization and stimulated proliferation in confluent cultures without apparent cell toxicity. Newborn rats injected with GA systemically also showed increased cell proliferation in different brain regions. Most of the proliferating cells displayed markers of immature astrocytes. Antioxidant iron porphyrins prevented both mitochondria dysfunction and increased in vitro and in vivo proliferation, suggesting a role of oxidative stress in inducing astrocytosis. Taken together, the data suggest that mitochondrial dysfunction induced by GA metabolites causes astrocytes to adopt a proliferative phenotype, which may underlie neuronal loss, white matter abnormalities and macrocephalia characteristics of GAI.

Topics: Amino Acid Metabolism, Inborn Errors; Animals; Animals, Newborn; Anthracenes; Antioxidants; Astrocytes; Brain; Brain Diseases, Metabolic, Inborn; Butadienes; Cell Count; Cell Proliferation; Cell Survival; Cells, Cultured; Glutarates; Immunohistochemistry; Membrane Potential, Mitochondrial; Mitochondria; Nitriles; Porphyrins; Rats; Rats, Sprague-Dawley

In addition to elevated concentrations of cytokines, patients with congestive heart failure (CHF) show endothelial dysfunction and increased plasma concentrations of adhesion molecules like intercellular adhesion molecule-1 (ICAM-1). Furthermore, the concentration of cardiotrophin-1 (CT-1)--a cytokine of the interleukin-6 superfamily--is increased in CHF. We tested the hypothesis whether CT-1 is able to induce ICAM-1 in human umbilical vein endothelial cells (HUVEC). Furthermore we examined the signalling mechanisms of CT-1 mediated ICAM-1 expression.. Confluent layers of HUVEC were incubated with increasing concentrations of CT-1 (5 to 100 ng/ml) for different periods. ICAM-1 mRNA was determined by real-time polymerase chain reaction (PCR) and ICAM-1 surface expression by fluorescence-activated cell sorter (FACS) analysis and soluble ICAM-1 (sICAM-1) in the culture supernatant by enzyme linked immunosorbent assay (ELISA). To clarify the signalling pathway of CT-1 induced ICAM-1 expression we used various inhibitors of possible signal transducing molecules, electromobility shift assay (EMSA) and Western blot analysis.. CT-1 induced ICAM-1 mRNA (1.8 +/- 0.8 fold increase compared to unstimulated cells after 6 hours) and protein (1.4 +/- 0.2 fold increase compared to unstimulated cells after 48 hours) in HUVEC in a time- and concentration-dependent manner. EMSA experiments show that CT-1 causes nuclear factor (NF) kappaB activation. Because parthenolide could inhibit CT-1 induced ICAM-1 expression NFkappaB activation is required in this pathway. CT-1 did not activate extracellular signal regulated kinases (ERK), c-Jun N-terminal kinase (JNK) and p38.. CT-1 is able to induce ICAM-1 in endothelial cells by NFkappaB activation. These results may explain in part elevated ICAM-1 concentrations in patients with CHF and endothelial dysfunction.

Topics: Anthracenes; Butadienes; Cells, Cultured; Cytokines; Electrophoretic Mobility Shift Assay; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Gene Expression; Humans; Intercellular Adhesion Molecule-1; JNK Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Polymerase Chain Reaction; Sesquiterpenes; Umbilical Veins

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

Topics: Animals; Anthracenes; Butadienes; CD40 Antigens; Cell Line; Cell Line, Tumor; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Flow Cytometry; Humans; Imidazoles; Immunoblotting; Leupeptins; Mitogen-Activated Protein Kinases; Monocytes; NF-kappa B; Nitriles; Phosphorylation; Polysaccharides, Bacterial; Protein Kinase Inhibitors; Pyridines; Signal Transduction; Sulfones; Time Factors; Toll-Like Receptor 4

Sjögren's syndrome and therapeutic radiation for head and neck cancers result in irreversible changes in the parenchyma of salivary glands, loss of acinar cells, prominence of duct cells, and fibrosis. To clarify mechanisms of salivary gland dysfunction, we identified a signaling pathway involved in the dedifferentiation of primary cultures of parotid acinar cells. We reported previously that the expression pattern of claudins changes during culture, is related to the three-dimensional organization of the cells, and reflects their ability to function as acinar cells. In this study, we found that this change of claudin expression is a process of dedifferentiation, because expression of other differentiation markers also changes during culture. The expression levels of claudins-4 and -6, cytokeratin 14, and vimentin are increased, and those of claudin-10, aquaporin 5, and amylase are decreased. Inhibitors of Src and p38 MAP kinases suppress these changes and increase the expression of acinar marker proteins. Differences in extracellular matrix components have no effect. Activation of p38 MAP kinase occurs during cell isolation from the parotid glands and is retained up to 6 h after the isolation. In contrast, activation of Src kinases does not increase during the cell isolation. The Src inhibitor PP1 suppresses the activation of p38 MAP kinase. Therefore, cellular stresses induced during cell isolation cause dedifferentiation and transition to duct-like cells through activation of p38 MAP kinase and constitutively active Src kinases.

Topics: Amylases; Animals; Anthracenes; Aquaporin 5; Butadienes; Cell Adhesion; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cell Shape; Cells, Cultured; Claudin-4; Claudins; Enzyme Activation; Extracellular Matrix; Imidazoles; Keratin-14; Male; Membrane Proteins; Nitriles; p38 Mitogen-Activated Protein Kinases; Parotid Gland; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Pyrimidines; Rats; Rats, Sprague-Dawley; Signal Transduction; src-Family Kinases; Tight Junctions; Time Factors; Vimentin

Inhibition of cytokines offers modest protection from injury in animal models of lung ischemia-reperfusion. Improved strategies would selectively inhibit the transcriptional activation response to oxidative stress. Mitogen-activated protein kinases (p38, c-jun N-terminal kinase, extracellular signal-regulated kinase) have been shown to be activated after oxidative stress and in animal models of acute inflammatory lung injury. We hypothesized that mitogen-activated protein kinase inhibition would block downstream transcriptional activation, providing robust protection from lung ischemia-reperfusion injury.. Experimental rats received inhibitors of p38, c-jun kinase, or extracellular signal-regulated kinase before in situ left lung ischemia-reperfusion. Immunohistochemistry localized cellular sites of mitogen-activated protein kinase activation. Several markers of lung injury were assessed. Enzyme-linked immunosorbent assay measured soluble cytokine and chemokine contents. Western blotting assessed mitogen-activated protein kinase phosphorylation. Electromobility shift assays measured transcription factor nuclear translocation.. Immunohistochemistry localized p38 and c-jun kinase activations in positive controls to alveolar macrophages. Extracellular signal-regulated kinase was activated in endothelial and epithelial cells. Animals treated with p38 or c-jun kinase inhibitor demonstrated significant reductions in transcription factor activation and markers of lung injury. Extracellular signal-regulated kinase inhibition was not protective. Western blotting confirmed inhibitor specificity.. Inhibition of p38 and c-jun kinase provided significant protection from injury. The alveolar macrophage appears to be the key coordinator of injury in response to oxidative stress. Therapeutically targeting specific cell population (macrophage) responses to oxidative stress has the potential benefit of reducing lung reperfusion injury severity while leaving host immune responses intact.

Topics: Animals; Anthracenes; Blotting, Western; Bronchoalveolar Lavage Fluid; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Female; Immunohistochemistry; Inflammation Mediators; Lung Diseases; Male; Mitogen-Activated Protein Kinases; Nitriles; Oxidative Stress; Peroxidase; Phosphorylation; Probability; Random Allocation; Rats; Rats, Sprague-Dawley; Reference Values; Reperfusion Injury; Sensitivity and Specificity

Matrix metalloproteinases (MMPs) play pivotal roles in inflammatory diseases including chronic periodontitis. The effects of Prevotella intermedia, a major periodontal pathogen, on MMP-9 production in primary human periodontal ligament (hPDL) cells were examined in the present study. MMP-9 mRNA expression was measured by semiquantitative reverse transcriptase PCR and its protein secretion was assayed by gelatin zymography. Prevotella intermedia ATCC 25611 supernatant time and dose-dependently induced MMP-9 expression. In contrast, Porphyromanas gingivalis ATCC 33277 supernatants, Escherichia coli lipopolysacchride and IL-1beta exhibited no stimulatory effects on MMP-9 production in hPDL cells. Mitogen-activated protein kinases [MAPK, including extracellular signal-related kinases (ERK), c-jun N-terminal kinases (JNK) and p38] inhibitors exerted no effect on the P. intermedia-induced MMP-9 production, indicating that P. intermedia induced MMP-9 production through an MAPK-independent pathway. Our results demonstrated that P. intermedia may contribute to periodontal tissue destruction during chronic periodontitis by inducing MMP-9 production in hPDL cells.

Topics: Adolescent; Anthracenes; Bacteroidaceae Infections; Butadienes; Cells, Cultured; Child; Culture Media, Conditioned; Electrophoresis, Polyacrylamide Gel; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Matrix Metalloproteinase 9; Nitriles; p38 Mitogen-Activated Protein Kinases; Periodontal Ligament; Porphyromonas gingivalis; Prevotella intermedia; Protein Kinase Inhibitors; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

Animal models show us that specific activation of the p38 mitogen-activated protein kinase (MAPK) may be a pivotal step in lidocaine neurotoxicity, but this has not been investigated in the case of two very widely used local anesthetics, bupivacaine and ropivacaine. We investigated the hypotheses that these drugs (A) are less neurotoxic than the prototype local anesthetic, lidocaine (B) are selectively toxic for subcategories of dorsal root ganglion neurons and (C) induce activation of either p38 MAPK or related enzymes, such as the c-jun terminal N-kinase (JNK) and extracellular signal-regulated kinase (ERK).. We incubated primary sensory neuron cultures with doses of lidocaine, bupivacaine, and ropivacaine equipotent at blocking sodium currents. Next, we sought to determine potential selectivity of bupivacaine and ropivacaine toxicity on neuron categories defined by immunohistochemical staining, or size. Subsequently, the involvement of p38 MAPK, JNK, and ERK was tested using enzyme-linked immunosorbent assays. Finally, the relevance of MAPK pathways in bupivacaine- and ropivacaine-induced neurotoxicity was determined by selectively inhibiting activity of p38 MAPK, JNK, and ERK.. We found that the neurotoxic potency of bupivacaine and ropivacaine is dose-dependent and similar in vitro, but is not selective for any of the investigated subgroups of neurons. Neurotoxicity of bupivacaine and ropivacaine was mediated, at least in part, by MAPKs. Specifically, we demonstrated the relevance of both p38 MAPK and JNK pathways for the neurotoxicity of bupivacaine and characterized the involvement of the p38 MAPK pathway in the neurotoxicity of ropivacaine.. Given equipotent doses, the neurotoxic potential of lidocaine does not appear to be significantly different from that of bupivacaine and ropivacaine in vitro. Moreover, bupivacaine and ropivacaine do not exert their neurotoxicity differently on specific subsets of dorsal root ganglion neurons. Their neurotoxic effects are brought about through the activation of specific MAPKs; the specific pharmacologic inhibition of these kinases attenuates toxicity in vitro.

Topics: Amides; Anesthetics, Local; Animals; Anthracenes; Bupivacaine; Butadienes; Cell Size; Cell Survival; Cells, Cultured; Cytoprotection; Dose-Response Relationship, Drug; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Ganglia, Spinal; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lidocaine; Membrane Potentials; Mitogen-Activated Protein Kinases; Neurons, Afferent; Nitriles; p38 Mitogen-Activated Protein Kinases; Phenotype; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Rats; Rats, Sprague-Dawley; Ropivacaine; Sodium Channels

Mechanical stimuli are important for maintaining organ structure and tissue function. To elucidate signalling pathways activated by mechanical stimuli, the contribution of mRNA stabilization to induction of egr-1 mRNA by medium flow was examined and the mechanisms responsible for stabilization were analysed. An early-response gene that encodes a transcription factor, egr-1, activates transcription of several genes in response to mechanical stimuli, and was therefore selected to resolve how early-induced signals are integrated and connected to subsequent response.. Mouse osteoblast-like MC3T3E1 cells were stably transfected with the chloramphenicol acetyltransferase (CAT) gene linked to the egr-1 promoter, and inductions of endogenous egr-1 and transfected CAT mRNA following medium flow were compared using real-time reverse transcriptase PCR. The mechanism of induction was examined using a transcription inhibitor and mitogen-activated protein (MAP) kinase inhibitors. Activation of MAP kinases by medium flow was investigated using western blotting.. Induction of egr-1 mRNA by medium flow was twofold higher than CAT mRNA induction. Induction of egr-1 mRNA was also observed in cells pre-treated with transcription inhibitor. The p38 inhibitor SB203580 enhanced induction of egr-1 mRNA by medium flow. Extracellular signal regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) were activated by medium flow.. A considerable part of egr-1 mRNA induction by medium flow may be due to mRNA stabilization. The p38 inhibitor SB203580 enhances induction.

Topics: Animals; Anthracenes; Butadienes; Cells, Cultured; Early Growth Response Protein 1; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Imidazoles; MAP Kinase Kinase 4; Mechanotransduction, Cellular; Mice; Nitriles; Osteoblasts; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Protein Kinase Inhibitors; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection

The components of Magnolia obovata are known to have many pharmacological activities. In this study, we investigated the effects of obovatol, a neolignan compound isolated from the leaves of M. obovata, on nitric oxide (NO) production and NF-kappaB activity in lipopolysaccharide (LPS)-activated RAW 264.7 cells. The results show that obovatol (1-5 microM) significantly inhibited LPS-induced NO production in a concentration-dependent manner (IC(50): 0.91 microM). Consistent with the inhibitory effect on NO production, obovatol inhibits the expression of inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, obovatol suppressed NF-kappaB (p50 and p65) translocation to the nucleus as well as IkappaB release resulting in the inhibition of the DNA binding activity of the NF-kappaB. Obovatol also inhibited c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signal, which are the most significantly involved signal in NO production and NF-kappaB activation. When the cells were treated with the combination of obovatol with U0126 (an ERK inhibitor) or SP600125 (a JNK inhibitor) as well as with SC-514 (an IKK2 inhibitor), much more inhibition of NO production was observed than that by obovatol alone. The present results suggest that obovatol has an inhibitory effect on NO production through the inhibition of NF-kappaB/MAPK activity, and thus can be used as an anti-inflammatory agent.

Topics: Animals; Anthracenes; Anti-Inflammatory Agents, Non-Steroidal; Biphenyl Compounds; Butadienes; Cell Line; Cyclooxygenase 2; Extracellular Signal-Regulated MAP Kinases; Lipopolysaccharides; Macrophages; Magnolia; MAP Kinase Kinase 4; Mice; Mitogen-Activated Protein Kinases; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Nitriles; Phenyl Ethers

Alpha-synuclein is an abundant neuronal protein that has been linked to both normal synaptic function and neurodegeneration--in particular, Parkinson's disease (PD). Uncovering mechanisms that control alpha-synuclein transcription is therefore critical for PD pathogenesis and synaptic function. We previously reported that in PC12 cells and primary neurons, alpha-synuclein is transcriptionally up-regulated after application of growth factors. In the current work we have characterized the pathway involved in this regulation in PC12 cells. The MAP/ERK pathway, and in particular Ras, is both sufficient and necessary for the NGF and basic fibroblast growth factor (bFGF) -mediated response. Significantly, response elements for this pathway, including a putative occult promoter, lie within intron 1, a hitherto unappreciated regulatory region of the gene that may be utilized in this or other settings. The PI3 kinase pathway is also involved in alpha-synuclein regulation, but response elements for this pathway appear to lie primarily outside of intron 1. These findings indicate that NGF- and bFGF-mediated signal transduction via the MAP/ERK and PI3 kinase pathways, and in part via regulatory regions within intron 1, may be involved in alpha-synuclein transcriptional regulation. Targeting of these pathways may serve to modulate alpha-synuclein so that it achieves desirable levels within neuronal cells.

Topics: alpha-Synuclein; Animals; Anthracenes; Blotting, Western; Butadienes; Chromones; Enzyme Inhibitors; Fibroblast Growth Factor 2; Gene Expression Regulation; Humans; Imidazoles; Introns; JNK Mitogen-Activated Protein Kinases; Models, Biological; Morpholines; Nerve Growth Factor; Nitriles; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pyridines; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that regulate key developmental processes, but are also overexpressed in many carcinomas. To assess whether BMPs would influence the three-dimensional architecture of epithelial structures, we took advantage of an in vitro model in which mammary epithelial cells form alveolar-like spherical cysts in collagen gels. We found that BMP-4 has a dramatic, biphasic effect on the organization of epithelial cysts. When added in the concentration range of 1-10 ng/ml, the cytokine abrogates lumen formation and induces the outgrowth of multiple invasive cord-like structures. At higher concentrations (20-100 ng/ml), BMP-4 additionally disrupts cell-cell adhesion, resulting in cyst disintegration and scattering of individual cells into the surrounding collagen matrix. The finding that BMP-4 subverts the ability of mammary epithelial cells to form polarized lumen-containing structures and endows them with invasive properties supports the involvement of this cytokine in the progression of breast cancer.

Topics: Animals; Anthracenes; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Butadienes; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Female; Humans; Imidazoles; Mammary Glands, Animal; Mice; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Nitriles

To examine the relative importance of tumour necrosis factor-receptor 1 (TNF-R1) and TNF-R2 and their signalling pathways for pro-inflammatory and pro-destructive features of early-passage synovial fibroblasts (SFB) from rheumatoid arthritis (RA) and osteoarthritis (OA).. Cells were stimulated with tumour necrosis factor (TNF)alpha or agonistic anti-TNF-R1/TNF-R2 monoclonal antibodies. Phosphorylation of p38, ERK and JNK kinases was assessed by western blot; proliferation by bromodesoxyuridine incorporation; interleukin (IL)6, IL8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase (MMP)-1 secretion by ELISA; and MMP-3 secretion by western blot. Functional assays were performed with or without inhibition of p38 (SB203580), ERK (U0126) or JNK (SP600125).. In RA- and OA-SFB, TNFalpha-induced phosphorylation of p38, ERK or JNK was exclusively mediated by TNF-R1. Reduction of proliferation and induction of IL6, IL8 and MMP-1 were solely mediated by TNF-R1, whereas PGE(2) and MMP-3 secretion was mediated by both TNF-Rs. In general, inhibition of ERK or JNK did not significantly alter the TNFalpha influence on these effector molecules. In contrast, inhibition of p38 reversed TNFalpha effects on proliferation and IL6/PGE(2) secretion (but not on IL8 and MMP-3 secretion). The above effects were comparable in RA- and OA-SFB, except that TNFalpha-induced MMP-1 secretion was reversed by p38 inhibition only in OA-SFB.. In early-passage RA/OA-SFB, activation of MAPK cascades and pro-inflammatory/pro-destructive features by TNFalpha is predominantly mediated by TNF-R1 and, for proliferation and IL6/PGE(2) secretion, exclusively regulated by p38. Strikingly, RA-SFB are insensitive to p38 inhibition of MMP-1 secretion. This indicates a resistance of RA-SFB to the inhibition of pro-destructive functions and suggests underlying structural/functional alterations of the p38 pathway, which may contribute to the pathogenesis or therapeutic sensitivity of RA, or both.

Topics: Anthracenes; Arthritis, Rheumatoid; Blotting, Western; Butadienes; Case-Control Studies; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Humans; Imidazoles; MAP Kinase Kinase 4; Matrix Metalloproteinase 1; Matrix Metalloproteinase Inhibitors; Nitriles; Osteoarthritis; p38 Mitogen-Activated Protein Kinases; Pyridines; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Signal Transduction; Statistics, Nonparametric; Synovial Membrane; Tumor Necrosis Factor-alpha

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme in the L-tryptophan-kynurenine pathway, which converts an essential amino acid, L-tryptophan, to N-formylkynurenine. The expression of IDO increases when inflammation is induced by wounding, infection or tumor growth. Although recent studies have suggested that IDO expression is up-regulated by IFN-gamma in various cell types and that the induction of IDO can also be mediated through an IFN-gamma-independent mechanism, these mechanisms still remain unknown. In this study, we investigated whether lipopolysaccharide (LPS) induces the expression of IDO through an IFN-gamma-mediated signaling pathway or not. IFN-gamma-induced expression of IDO expression was inhibited only by JAK inhibitor I. However, LPS-induced expression of IDO was inhibited by LY294002 and SP600125 but not by JAK inhibitor I, SB203580, or U0126. These findings clearly indicate that LPS can induce the IDO expression via an IFN-gamma-independent mechanism and PI3 kinase and JNK in the LPS-induced pathway leading to IDO expression.

Topics: Animals; Anthracenes; Bone Marrow Cells; Butadienes; Chromones; Dendritic Cells; Gene Expression; Gene Expression Regulation, Enzymologic; Imidazoles; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interferon-gamma; Janus Kinase 1; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Male; Mice; Mice, Inbred Strains; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Pyridines

Bacillus anthracis, the causative agent of inhalational anthrax, enters a host through the pulmonary system before dissemination. We have previously shown that human alveolar macrophages participate in the initial innate immune response to B. anthracis spores through cell signal-mediated cytokine release. We proposed that the lung epithelia also participate in the innate immune response to this pathogen, and we have developed a human lung slice model to study this process. Exposure of our model to B. anthracis (Sterne) spores rapidly activated the mitogen-activated protein kinase signaling pathways ERK, p38, and JNK. In addition, an RNase protection assay showed induction of mRNA of several cytokines and chemokines. This finding was reflected at the translational level by protein peak increases of 3-, 25-, 9-, 34-, and 5-fold for interleukin-6 (IL-6), tumor necrosis factor alpha, IL-8, macrophage inflammatory protein 1alpha/beta, and monocyte chemoattractant protein 1, respectively, as determined by an enzyme-linked immunosorbent assay. Inhibition of individual pathways by UO126, SP600125, and SB0203580 decreased induction of chemokines and cytokines by spores, but this depended on the pathways inhibited and the cytokines and chemokines induced. Combining all three inhibitors reduced induction of all cytokines and chemokines tested to background levels. An immunohistochemistry analysis of IL-6 and IL-8 revealed that alveolar epithelial cells and macrophages and a few interstitial cells are the source of the cytokines and chemokines. Taken together, these data showed the activation of the pulmonary epithelium in response to B. anthracis spore exposure. Thus, the lung epithelia actively participate in the innate immune response to B. anthracis infection through cell signal-mediated elaboration of cytokines and chemokines.

Topics: Anthracenes; Anthrax; Bacillus anthracis; Butadienes; Chemokines; Cytokines; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Gene Expression Regulation; Humans; Imidazoles; Immunity, Innate; Immunohistochemistry; In Vitro Techniques; Lung; Macrophages, Alveolar; MAP Kinase Signaling System; Microscopy, Fluorescence; Nitriles; Pulmonary Alveoli; Pyridines; RNA, Messenger; Spores, Bacterial

Elevated exposure to manganese is known to cause neurodegeneration in the basal ganglia and to induce movement abnormalities called manganism. However, the underlying mechanism of action is not fully understood. Activation of the resident immune cells in the brain, microglia that release a variety of neurotoxic factors, has been implicated to contribute to neurodegeneration. Of the various neurotoxic factors released by activated microglia, reactive oxygen species such as superoxide and hydrogen peroxide are particularly detrimental to the survival of the oxidative damage-prone neurons. In this study, we report that exposure of rat microglia to manganese chloride (MnCl(2)) resulted in a time- and concentration-dependent release of hydrogen peroxide (H(2)O(2)). The MnCl(2)-stimulated microglial H(2)O(2) release was sensitive to inhibitors of mitogen-activated protein kinases (MAPK) but not that of NADPH oxidase. MnCl(2)-induced a rapid activation of the extracellular signal-regulated kinase (ERK) and p38-MAPK in microglia that appeared to precede the MnCl(2)-induced H(2)O(2) release, suggesting that ERK and p38-MAPK influenced the MnCl(2)-induced H(2)O(2) release in microglia. In summary, these results demonstrate that manganese chloride is capable of activating microglia to release ROS and MAPK may, in part, be key regulators of the process. These findings may shed significant light on the potential role of microglia in the manganese-induced neurotoxicity.

Topics: Animals; Animals, Newborn; Anthracenes; Butadienes; Cell Line; Cells, Cultured; Chlorides; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hydrogen Peroxide; Imidazoles; Manganese Compounds; Microglia; NADPH Oxidases; Nitriles; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Rats, Inbred F344; Time Factors

Mutations in Ras family genes are rare in malignant mesothelioma. The role of activation of the Ras signaling pathway in the pathogenesis of mesothelioma is not clear.. We studied the activation status of the Ras pathway and the status of other Ras-associated kinases in a panel of human mesothelioma cell lines. In addition, we tested the effect of inhibition of several kinase pathways on mesothelioma cell proliferation. The potential role of kinase signaling on the regulation of cap-dependent translation was also studied.. In general, Ras-guanosine triphosphate (GTP) was higher in mesothelioma cell lines when compared with a nontransformed mesothelial cell line (LP9). Furthermore, known Ras effectors such as extracellular-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase were found to be active in most of the mesothelioma cell lines tested. Exposure to specific inhibitors of extracellular-regulated kinase 1/2 (U0126) and c-Jun N-terminal kinase (SP600125) significantly decreased the proliferation of H2596 and H2373 cells compared with mock-treated cells. SP600125-mediated c-Jun N-terminal kinase inhibition, but not extracellular-regulated kinase 1/2 inhibition, resulted in a decrease in phosphorylation of 4E-BP1, consequently decreasing cap-dependent activation.. These experiments provide a rationale for targeting Ras and associated signaling pathways in mesothelioma and also suggest cap-dependent translation as one mechanism by which Ras induces proliferation in this disease.

Topics: Adaptor Proteins, Signal Transducing; Anthracenes; Apoptosis; Blotting, Western; Butadienes; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; DNA, Neoplasm; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; Mesothelioma; Mitogen-Activated Protein Kinase 3; Mutation; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphoproteins; Phosphorylation; Polymerase Chain Reaction; Repressor Proteins; Signal Transduction

Cyclooxygenase-2 (COX-2) is induced by hypoxic stimuli and is also involved in the process of angiogenesis. We previously demonstrated that vascular endothelial growth factor (VEGF) is one of the principal factors produced by hypoxic myocytes and is responsible for the induction of COX-2 expression in endothelial cells. Yet the signaling pathways by which VEGF modulates COX-2 gene expression are still less well defined. We therefore examined the regulation of VEGF-induced COX-2 expression by the mitogen-activated protein kinase (MAPK) family in endothelial cells.. Human umbilical vascular endothelial cells (HUVECs) were incubated with U0126 (ERK1/2 inhibitor, 10 microM), SB203580 (p38 inhibitor, 20 microM), and SP600125 (JNK inhibitor, 20 microM), as well as the COX-2 selective inhibitor, NS398, for 1 h before treating with VEGF (20 ng/ml). COX-2 expression induced by VEGF at both mRNA and protein levels was significantly inhibited by selective p38 and JNK inhibitors but not by the ERK1/2 inhibitor. The phosphorylation of p38 and JNK kinases was observed as early as 5 min in HUVECs after VEGF stimulation. Furthermore, the biological significance of the COX-2 gene in endothelial cells was examined by over-expressing or knocking down COX-2 gene expression. (3)H-Thymidine incorporation and Matrigel techniques were used to determine cell proliferation and vascular structure formation. VEGF-induced cell proliferation was significantly reduced when HUVECs were either pre-treated with NS398 (21.52+/-3.6%) or transfected with COX-2 siRNA (34.12+/-5.81%). In contrast, in HUVECs with over-expression of COX-2, VEGF-induced cell proliferation was increased 42.56+/-7.69%. Moreover, the formation of vascular structure assayed by Matrigel demonstrated that VEGF-induced vascular structure formation was accelerated in COX-2 over-expressing cells but attenuated in COX-2 siRNA-transfected cells.. COX-2 plays an important role in VEGF-induced angiogenesis via p38 and JNK kinase activation pathways. These findings suggest that the cardioprotective role of COX-2 may be, at least in part, through its angiogenic activity.

Topics: Anthracenes; Butadienes; Cells, Cultured; Cyclooxygenase 2; Endothelial Cells; Endothelium, Vascular; Gene Deletion; Humans; Imidazoles; MAP Kinase Kinase 4; Membrane Proteins; Neovascularization, Physiologic; Nitriles; Nitrobenzenes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; RNA, Small Interfering; Signal Transduction; Sulfonamides; Vascular Endothelial Growth Factor A

Bacillus anthracis must overcome host innate immune defences to establish a systemic anthrax infection. This is facilitated in part by lethal toxin (LT), a secreted virulence factor that consists of a cell-binding moiety, protective antigen (PA), and an enzymatic moiety, lethal factor (LF). PA binds cells through protein receptors and mediates the delivery of LF to the cytosol. LF is a protease that cleaves amino-terminal fragments from mitogen-activated protein kinase kinases (MAPKKs), preventing phosphorylation of their downstream targets. Here we report that LT reduces the amount of interleukin (IL)-8 produced and secreted by human endothelial cells. The reduction of IL-8 levels by LT was not attributable to reduced expression from the IL-8 promoter, but resulted from destabilization of IL-8 mRNA. Destabilization by LT was mediated through the 3' untranslated region of the IL-8 transcript and could be mimicked by pharmacological inhibitors of MAPK pathways. LT diminished the induction of IL-8 mRNA and protein by lipopolysaccharide, indicating that the toxin can impair the ability of these cells to initiate an immune response. Destabilization of a cytokine transcript represents a new interference strategy used by either a bacterial or viral pathogen to reduce cytokine expression and may help B. anthracis to evade host immune defences.

Topics: 3' Untranslated Regions; Anthracenes; Antigens, Bacterial; Bacillus anthracis; Bacterial Toxins; Butadienes; Cells, Cultured; Endothelial Cells; Humans; Imidazoles; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Mitogen-Activated Protein Kinases; Nitriles; Promoter Regions, Genetic; Pyridines; RNA Stability; RNA, Messenger; Signal Transduction

Little is known about the pathogenesis of apoptosis caused in cardiac tissues by periodontitis pathogens. The purpose of this study was to determine the related effect of periodontal pathogen Porphyromonas gingivalis on cardiac cell apoptosis.. DNA fragmentation, nuclear condensation and activated apoptotic caspases were measured by agarose gel electrophoresis, nuclear DAPI (4',6-diamidine-2-phenylindole dihydrochloride) stain and western blotting analysis following the surrounding medium of P. gingivalis and/or pre-administration of SB203580 (p38 inhibitor), U0126 [mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor], LY294002 [phosphoinositide 3-kinase (PI3K) inhibitor], cyclosporine A (CsA: calcineurin inhibitor), and Sp600125 [c-Jun N-terminal kinase (JNK) inhibitor] in cultured cardiac H9c2 cells.. The surrounding medium of periodontal pathogen P. gingivalis increased DNA fragmentation, nuclear condensation and the activated apoptotic caspase-3, -8, and -9 proteins in H9c2 cells. DNA fragmentation and nuclear condensation of H9c2 cells treated with P. gingivalis medium were completely blocked by SB203580 plus U0126 and were decreased after pre-administration of SB203580 only, U0126 only, LY294002, CsA, but were increased by Sp600125.. Our findings suggest that the development of cardiac cell apoptosis can be directly induced by P. gingivalis medium. Porphyromonas gingivalis-related H9c2 cell apoptosis was mainly co-activated by p38 and ERK pathways and may be involved in death receptor-dependent (caspase 8) and mitochondria (caspase 9)-dependent apoptotic pathways. Porphyromonas gingivalis-related cardiac cell apoptosis was also partially mediated by PI3K or calcineurin signaling pathways, whereas the JNK pathway might play a protective role in P. gingivalis-related cardiac cell apoptosis.

Topics: Animals; Anthracenes; Apoptosis; Butadienes; Calcineurin Inhibitors; Caspases; Cell Nucleus; Chromones; Cyclosporine; DNA Fragmentation; Enzyme Inhibitors; Fluorescent Dyes; Imidazoles; Indoles; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Morpholines; Myocardium; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphoinositide-3 Kinase Inhibitors; Porphyromonas gingivalis; Pyridines; Rats

Trimidox (3,4,5-trihydroxybenzamidoxime) has been shown to reduce the activity of ribonucleotide reductase accompanied by growth inhibition and the differentiation of mammalian cells. Here we examine the induction of apoptosis by trimidox in several human leukaemia cell lines, focusing on the release of cytochrome c and the activation of caspase proteases in the human B cell line NALM-6. Induction of apoptosis by trimidox (300 microM) was detected in NALM-6, HL-60 (premyelocytic leukaemia cells), MOLT-4 (an acute lymphoblastic leukaemia cells), Jurkat (a T-cell leukaemia cells), U937 (expressing many monocyte-like characteristics), and K562 (erythroleukaemia). NALM-6 was most affected by trimidox among leukaemia cells; therefore, we employed NALM-6 cells in the subsequent experiments. The cells showed a time-dependent increase in DNA damage after trimidox (250 microM) treatment. A significant increase in the amount of cytochrome c release was detected after treatment with trimidox. Bcl-2 and Bax protein expressions were not changed by trimidox. Caspase-3 and -9 were activated by incubation with trimidox, whereas caspase-8 was not. Furthermore, trimidox-induced apoptosis was prevented by a broad-spectrum caspase inhibitor, a caspase-3, and a caspase-9 inhibitor, but not by a caspase-8 inhibitor. Inhibition of c-Jun NH2-terminal kinase (JNK) by SP600125 appreciably protected cells from trimidox-induced apoptosis, but no effect inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580. In contrast, extracellular signal-regulated kinase (ERK) inhibitors U0126 and PD98059 strongly potentiated the apoptotic effect of trimidox. This report shows that the induction of apoptosis by trimidox occurs through a cytochrome c-dependent pathway, which sequentially activates caspase-3 and caspase-9.

Topics: Anthracenes; Apoptosis; Benzamidines; Butadienes; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia, B-Cell; Nitriles; Oligopeptides; Ribonucleotide Reductases; Signal Transduction; Time Factors

Hemin is an oxidant that accumulates in intracranial hematomas. Its neurotoxicity is increased by its breakdown, which is catalyzed by the heme oxygenase (HO) enzymes. In this study we tested the hypothesis that inhibiting signaling events mediating HO-1 induction would protect cultured cortical neurons from hemin. A fivefold increase in HO-1 expression was observed in mixed neuron-astrocyte cultures 4h after hemin exposure. This was markedly reduced by the ERK pathway inhibitor U0126. The JNK inhibitor SP600125 had a weak but statistically significant effect, while the p38 inhibitor SB239063 was ineffective. Hemin neurotoxicity, as assessed by LDH release, propidium iodide staining, and malondialdehyde assay, was also prevented by U0126 but not by SB239063; SP600125 had little or no effect. Consistent with reduced iron release, ferritin expression was also attenuated by U0126, while cell hemin accumulation was increased. These results suggest that targeting the ERK pathway may prevent HO-1 induction in response to hemin, and reduce neuronal injury.

Topics: Animals; Anthracenes; Astrocytes; Butadienes; Cell Death; Cells, Cultured; Coculture Techniques; Culture Media, Serum-Free; Embryo, Mammalian; Heme; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hemin; Imidazoles; JNK Mitogen-Activated Protein Kinases; Membrane Proteins; Mice; Mitogen-Activated Protein Kinases; Neurons; Nitriles; Pyrimidines; Signal Transduction

The signaling pathway that is initiated by binding of epidermal growth factor receptor (EGFR) and results in sustained signaling through PI3K plays an important role in a tumor's response to ionizing radiation. The current in vitro study explored both the effects of ZD1839 (Iressa), a highly selective EGFR tyrosine kinase inhibitor, as a radiosensitiser for bile duct carcinoma cell lines and ZD1839's general effects on cell growth in the same two lines. Secondly, we ensured suppression of radiation-induced phosphorylation of EGFR by ZD1839 using an immunoprecipitation technique. Furthermore, we examined radiation-induced phosphorylation of ERK, p38, JNK, and AKT with or without inhibitor with use of Western blot techniques and performed clonogenic assays to confirm radiosensitivity in the presence of a drug. ZD1839 inhibited cell growth of both cell lines and suppressed radiation-induced phosphorylation of EGFR. After exposure to radiation, there was an increase in phosphorylation of AKT as shown by Western blot. Treatment with either ZD1839 or LY294002 (the latter, a PI3K inhibitor) suppressed phosphorylation of AKT by Western blot. Both ZD1839 and LY294002 significantly suppressed colony formation by clonogenic assay; however, U0126 (a MEK1/2 inhibitor), SB203580 (a p38 inhibitor), and SP600125 (a JNK inhibitor) had no effect on colony formation. These results suggest that AKT may be a useful target molecule for enhancement of radiotherapy effect and that ZD1839 may have an important role in combination with radiotherapy for patients with bile duct carcinoma.

Topics: Anthracenes; Bile Duct Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; Clone Cells; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Gefitinib; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Oncogene Protein v-akt; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Quinazolines; Radiation-Sensitizing Agents

Roles of mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-induced production of histamine in the mouse macrophage-like cell line RAW 264 were analyzed. Incubation of RAW 264 cells in the presence of LPS increased histamine levels in the conditioned medium in a concentration- and time-dependent manner. The levels of histidine decarboxylase (HDC) mRNA and the 74-kDa HDC protein were also increased at 4 to 8 h and 8 to 12 h, respectively. LPS elicited the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and c-Jun N-terminal kinase (JNK). The MAP kinase-Erk kinase 1 inhibitor U0126 (0.1-10 microM) suppressed the LPS-induced phosphorylation of p44/42 MAP kinase, and inhibited the LPS-induced production of histamine and expression of the HDC mRNA and 74-kDa HDC protein in a concentration-dependent manner. The JNK inhibitor SP600125 (3-30 microM) suppressed the LPS-induced phosphorylation of c-Jun, and inhibited the LPS-induced production of histamine and expression of the HDC mRNA and 74-kDa protein in a concentration-dependent manner. Combined treatment with U0126 (0.3 microM) and SP600125 (10 microM) inhibited the LPS-induced production of histamine additively. The p38 MAP kinase inhibitor SB203580 (0.1-10 microM) partially inhibited the LPS-induced production of histamine. These findings suggest that LPS increases histamine production in RAW 264 cells by inducing the expression of the 74-kDa HDC protein, and that the LPS-induced expression of HDC is up-regulated at the transcriptional level by MAP kinases, especially p44 MAP kinase and JNK.

Topics: Animals; Anthracenes; Butadienes; Cell Line; Extracellular Signal-Regulated MAP Kinases; Histamine; Histidine Decarboxylase; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Macrophages; Mice; Mitogen-Activated Protein Kinases; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; RNA, Messenger

Our previous study have demonstrated that the expression of transforming growth factor-beta1 (TGF-beta1) by HKC could be up-regulated by aldosterone (ALDO) in vitro. The present study was designed to evaluate the role of MAPK/ERK1/2 phosphorylation in mediating the synthesis of TGF-beta1 in renal tubular epithelial cells that was activated by aldosterone.. The following tests were performed in vitro: (1) HKC were pretreated with different concentrations of specific ERK1/2, JNK and P38 MAPK pathway inhibitors for 4h, then HKC were stimulated with 10(-7) mol/L ALDO for 48 h, finally enzyme-linked immunosorbent assay (ELISA) were performed to detect TGF-beta1 expression; (2) HKC were stimulated with ALDO at different concentrations and times, then western blot assay was performed to detect the expression of phosphorylated and total ERK1/2 in the cell lysate of HKC. (3) HKC which were co-stimulated with 10(-7) mol/L ALDO and different concentrations of spironolactone or specific glucocorticoid hormone receptor inhibitor RU486 for 30min, then western blot assay was performed to detect the expression of phosphorylated and total ERK1/2 in the cell lysate of HKC.. (1) the production in 15 and 25 micromol/L U0126 incubated groups was (87 +/- 11) pg/ml and (75 +/- 19) pg/ml respectively, which was significantly decreased compared with that in 10(-7) mol/L ALDO incubated group (P < 0.05), however, the amount of TGF-beta1 in these groups were still significant higher than that in the control group (P < 0.05). The production of TGF-beta1 in the groups which were incubated with SP600125 and SB203580 did not appear significant decrease compared with that in 10(-7) mol/L ALDO incubated group (P > 0.05), the production of TGF-beta1 in these groups was also significant higher than that in the control group (P < 0.05). (2) The Phos/Total ERK1/2 ratio was increased in a dose-dependent manner. After HKC were stimulated with 10(-9) - 10(-7) mol/L ALDO for 30 min. Phos/TotalERK1/2 ratio was 0.67 +/- 0.06 and 0.80 +/- 0.05 respectively, which was significantly increased (vs 0 mol/L ALDO, P < 0.05 or 0.01). The expression of Phos/Total ERK1/2 ratio also had a positive correlations with the production of TGF-beta1 (R = 0.793, P < 0.01). With 10(-7) mol/L ALDO stimulated at different times, the Phos/Total ERK1/2 ratio was also significantly increased (vs 0 h, P < 0.05 or 0.01), which began to be increased and reached the peak at 15 min, the relatively ratio was 0.84 +/- 0.06, and waned till 240 min, and returned to normal level at 360 min. (3) After co-stimulated with 10(-7) mol/L ALDO and different concentration of spironolactone, Phos/TotalERK1/2 ratio was significantly decreased along with the concentrations of spironolactone, the relatively ratio of in 10(-9) - 10(-7) was 0.62 +/- 0.08 and 0.60 +/- 0.04 separately (vs 0 mol/L ALDO, or 0.01), but they were still significantly higher than that in control group (P < 0.05). The Phos/TotalERK1/2 ratio was not changed significantly along with the concentrations of RU486 (P > 0.05).. The effect of aldosterone in up-regulating the expression of TGF-beta1 in HKC is mediated, at least in part, by MAPK/ERK1/2 pathway. Aldosterone may exert this effect on the conditions of binding to the mineralocorticoid receptor first.

Topics: Aldosterone; Anthracenes; Blotting, Western; Butadienes; Cells, Cultured; Dose-Response Relationship, Drug; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; Kidney Tubules, Proximal; Mifepristone; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Pyridines; Signal Transduction; Time Factors; Transforming Growth Factor beta1

Reactive microglia are thought to play a role in the pathogenesis of Alzheimer's disease (AD) and are localized to the senile plaques that are associated with cognitive decline. The beta-amyloid precursor protein (betaAPP) is over-expressed in the dystrophic neurites near such plaques, and secreted forms of betaAPP (sAPPalpha) activate inflammatory responses in microglia. To characterize the mechanisms by which sAPPalpha activates microglia, we assayed its effects on MAP kinases, including c-Jun N-terminal kinases (JNK), extracellular signal-regulated protein kinases (ERK), and p38-MAPK. sAPPalpha was found to rapidly activate JNKs, ERKs and p38-MAPK in a dose-dependent manner. The JNK inhibitor SP600125 and the p38 inhibitor SB203580 independently reduced both nitrite accumulation and induction of inflammatory nitric oxide synthase (iNOS). By contrast, inhibition of the ERK pathway with U0126 did not appreciably affect either outcome measure. These findings suggest that sAPP activates the ERK, JNK and p38 classes of MAP kinases but that only JNK and p38-MAPK are critical for activation of microglia by sAPPalpha, a process that compromises neuronal function and survival.

Topics: Amyloid beta-Protein Precursor; Animals; Animals, Newborn; Anthracenes; Blotting, Western; Butadienes; Cells, Cultured; Dose-Response Relationship, Drug; Drug Combinations; Extracellular Signal-Regulated MAP Kinases; Imidazoles; JNK Mitogen-Activated Protein Kinases; Microglia; Nitriles; Nitrites; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Time Factors

We have examined the significance of the activation of c-Jun N-terminal kinase (JNK) and p42/44 mitogen-activated protein kinase (MAPK) by ethanol and acetaldehyde in rat hepatocyte apoptosis. Acetaldehyde induced rapid and transient (15 min) activation of p42/44 MAPK followed by activation of JNK, which remained above control up to 1 h. Ethanol activated JNK for up to 4 h. Both ethanol and acetaldehyde caused apoptosis as determined by DNA fragmentation, caspase-3 activation and 2'[4-ethoxyphenyl]-5-[4-methyl-piperazinyl]-2,5'-bi-1H-benzimidazole (Hoechst 33342) staining. Ethanol-induced apoptosis was blocked by JNK inhibitor 1,9-pyrazoloanthrone (SP600125), indicating that JNK activation is pro-apoptotic. In contrast, acetaldehyde-induced apoptosis was not suppressed by this inhibitor. In fact, SP600125 potentiated acetaldehyde-induced apoptosis, suggesting that JNK activation is anti-apoptotic. Inhibition of p42/44 MAPK by MAPK kinase (MKK1) inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), potentiated apoptosis by acetaldehyde or ethanol, suggesting anti-apoptotic role of p42/44 MAPK. The activation of JNK by ethanol or acetaldehyde was insensitive to the genistein (tyrosine kinase inhibitor), GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide, protein kinase C [PKC] inhibitor) and N-acetylcysteine (N-AC) (antioxidant), whereas p42/44 MAPK activation by acetaldehyde was inhibited by genistein and GF109203X. Furthermore, p42/44 MAPK activation is not necessary for the JNK activation. In summary, transient activation of JNK by acetaldehyde is anti-apoptotic, whereas sustained activation of JNK by ethanol is pro-apoptotic. The activation of p42/44 MAPK appears to be anti-apoptotic for both ethanol and acetaldehyde. Thus, JNK activation by ethanol and acetaldehyde can be both pro- and anti-apoptotic in hepatocytes.

Topics: Acetaldehyde; Acetylcysteine; Animals; Anthracenes; Apoptosis; Blotting, Western; Butadienes; Buthionine Sulfoximine; Caspase 3; Caspases; Cell Nucleus; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Ethanol; Genistein; Hepatocytes; Hydrogen Peroxide; Indoles; JNK Mitogen-Activated Protein Kinases; Male; Maleates; Maleimides; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Time Factors

Mast cell chymase is known to induce eosinophil migration in vivo and in vitro. In the present study, we investigated possible involvement of mitogen-activated protein (MAP) kinases; extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, in the chymase-induced eosinophil migration. Human chymase induced a rapid phosphorylation of ERK1/2 and p38 in human eosinophilic leukemia EoL-1 cells, while no phosphorylation was detected in JNK. The chymase-induced phosphorylation of ERK and p38 was inhibited by pertussis toxin. Similar results were obtained in the experiments using mouse chymase and eosinophils. U0126 (the inhibitor for MAP/ERK kinase) suppressed chymase-induced migration of EoL-1 cells and mouse eosinophils. However, SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) showed little effect on the migration. It is suggested therefore that chymase activates ERK and p38 probably through G-protein-coupled receptor, and that ERK but not p38 cascade may have a crucial role in chymase-induced migration of eosinophils.

Topics: Animals; Anthracenes; Blotting, Western; Butadienes; Butyric Acid; Cell Line, Tumor; Cell Movement; Chemotaxis; Chymases; Enzyme Inhibitors; Eosinophils; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Mast Cells; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinase 3; Nitriles; p38 Mitogen-Activated Protein Kinases; Pertussis Toxin; Phosphorylation; Pyridines; Receptors, G-Protein-Coupled; Serine Endopeptidases; Signal Transduction; Time Factors

Oxygen enhancement of tumor radiosensitivity is attributed to DNA damage by reactive oxygen species. The mechanism remains unclear but may involve mitochondria as major sources of oxygen and nitrogen radicals as well as central effectors of energy homeostasis and apoptosis. Here we used dihydrorhodamine and 2',7'-dichlorodihydrofluorescein to compare mitochondrial and total cell generation, respectively, of reactive oxygen or nitrogen species in cells irradiated at 5 Gy. Irradiation in the presence of oxygen selectively stimulated mitochondrial radical production in HeLa and MeWo cells, but in MCF7 cells radical production was more generalized. In all three cell lines oxygen impaired cell proliferation as measured by resazurin reduction 7 days after irradiation. Antioxidants N-acetylcysteine, ascorbic acid, and melatonin largely prevented dye oxidation during normoxic irradiation yet had no effect on oxygen-dependent irradiation injury. However, NO synthase inhibitor N(G)-monomethyl-L-arginine protected HeLa and MCF7 though not MeWo cells, consistent with their different levels of constitutive NO generation. SB203580 inhibition of p38 MAPK appreciably protected HeLa and marginally protected MCF7 cells against oxygen-dependent irradiation injury, while the less specific JNK/SAPK inhibitor SP600125 and ERK inhibitor U0126 had no effect. None of the inhibitors affected MeWo radiosensitivity. Therefore oxygen-enhanced radiosensitivity in these tumor cell lines does not depend on extensive production of oxygen radicals and is cell-type dependent. NO mediates oxygen-dependent injury in HeLa and MCF7 cells, by p38-dependent and MAPK-independent mechanisms, respectively. In MeWo cells this oxygen-enhanced radiosensitivity is independent of both NO and MAPK signaling.

Topics: Acetylcysteine; Anthracenes; Antioxidants; Apoptosis; Ascorbic Acid; Butadienes; Cell Line, Tumor; Cell Proliferation; DNA Damage; Enzyme Inhibitors; Fluoresceins; HeLa Cells; Humans; Imidazoles; MAP Kinase Signaling System; Melatonin; Mitochondria; Nitriles; Nitrites; Oxazines; Oxygen; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Radiation Tolerance; Rhodamines; Spectrometry, Fluorescence; Xanthenes

Ethanol treatment increases gene expression in the liver through mechanisms that are not clearly understood. Histone acetylation has been shown to induce transcriptional activation. We have investigated the characteristics and mechanisms of ethanol-induced histone H3 acetylation in rat hepatocytes. Immunocytochemical and immunoblot analysis revealed that ethanol treatment significantly increased H3 acetylation at Lys9 with negligible effects at Lys14, -18, and -23. Acute in vivo administration of alcohol in rats produced the same results as in vitro observations. Nuclear extracts from ethanol-treated hepatocytes increased acetylation in H3 peptide to a greater extent than extracts from untreated cells, suggesting that ethanol either increased the expression level or the specific activity of histone acetyltransferases (HAT). Use of different H3 peptides indicated that ethanol selectively modulated HAT(s) targeting H3-Lys9. Treatment with acetate, an ethanol metabolite, also increased acetylation of H3-Lys9 and modulated HAT(s) in the same manner as ethanol, suggesting that acetate mediates the ethanol-induced effect on HAT. Inhibitors of MEK (U0126) and JNK (SP600125), but not p38 MAPK inhibitor (SB203580), suppressed ethanol-induced H3 acetylation. However, U0126 and SP600125 did not significantly affect ethanol-induced effect on HAT, suggesting that ERK and JNK regulate histone acetylation through a separate pathway(s) that does not involve modulation of HAT. Chromatin immunoprecipitation assay demonstrated that ethanol treatment increased the association of the class I alcohol dehydrogenase (ADH I) gene with acetylated H3-Lys9. These data provide first evidence that ethanol increases acetylation of H3-Lys9 through modulation of HAT(s) and that histone acetylation may underlie the mechanism for ethanol-induced ADH I gene expression.

Topics: Acetylation; Alcohol Dehydrogenase; Animals; Anthracenes; Butadienes; Ethanol; Gene Expression Regulation, Enzymologic; Hepatocytes; Histone Acetyltransferases; Histones; Imidazoles; Male; MAP Kinase Signaling System; Nitriles; Pyridines; Rats; Rats, Sprague-Dawley

Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Extensive studies have indicated that anthocyanins have strong antioxidant activities. To investigate the mechanism of anthocyanidins as an anticancer food source, six kinds of anthocyanidins representing the aglycons of most anthocyanins, were used to examine their effects on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Of the six anthocyanins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 transactivation, suggesting that the ortho-dihydroxyphenyl may contribute to the inhibitory action. Delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in the extracellular signal-regulated protein kinase (ERK) pathway at early times and the c-Jun N-terminal kinase (JNK) signaling pathway at later times. p38 kinase was not inhibited by delphinidin. Furthermore, two mitogen-activated protein kinase (MAPK) specific inhibitors (SP600125 for JNK and UO126 for ERK) could specifically block the activation of JNK and ERK and cell transformation. Those results demonstrate that anthocyanidins contribute to the inhibition of tumorigenesis by blocking activation of the MAPK pathway. These findings provide the first molecular basis for the anticarcinogenic action of anthocyanidins.

Topics: Animals; Anthocyanins; Anthracenes; Anticarcinogenic Agents; Butadienes; Cell Transformation, Neoplastic; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Structure-Activity Relationship; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Transcription Factor AP-1

Genes encoding numerous proto-oncogenes and cytokines, as well as a number of G-protein coupled receptors, are regulated post-transcriptionally at the level of mRNA stability. A common feature of all of these genes is the presence of A + U-rich elements (AREs) within their 3' untranslated regions. We, and others, have demonstrated previously that mRNAs encoding beta-adrenergic receptors (beta-ARs) are destabilized by agonist stimulation of the beta-AR/Galphas/adenylylcyclase pathway. However, in addition to PK-A, beta-ARs can also activate or inhibit mitogen activated kinase (MAPK) cascades, in a cell-type dependent basis. Recent evidence points to an important role for MAPKs in regulating the turnover of cytokine mRNAs, such as TNFalpha. We hypothesized that activation of MAPK's may also regulate beta-AR mRNA stability. The studies conducted herein demonstrate that generalized stimulation of MAPKs (JNK, p38) with anisomycin resulted in marked stabilization of beta-AR mRNA. Reciprocally, selective inhibition of JNK with SP600125 significantly decreased beta-AR mRNA half-life. Similarly, inhibition of the MEK/ERK pathway with either PD98059 or U0126 decreased beta-AR mRNA stability substantially. However, inhibition of p38 MAPK with SB203580 produced destabilization of beta-AR mRNA only at higher, non pharmacologically selective concentrations. In contrast to their effects on several other ARE containing mRNAs, inhibition of tyrosine kinases by genistein or PI3K by wortmannin, had no detectable effect on beta-AR mRNA stability. In summary, these results demonstrate for the first time that modulation of MAPK pathways can bi-directionally influence beta-AR mRNA stability.

Topics: 3' Untranslated Regions; Animals; Anisomycin; Anthracenes; Butadienes; Cell Line; Cricetinae; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Heterogeneous-Nuclear Ribonucleoproteins; Imidazoles; Mitogen-Activated Protein Kinase Kinases; Nitriles; Nucleic Acid Synthesis Inhibitors; Phosphatidylinositol 3-Kinases; Pyridines; Receptors, Adrenergic, beta; RNA Stability; Signal Transduction

The natural hormone 17beta-estradiol (17beta-E2) is known to induce tumor angiogenesis in various target organs by activating positive regulators of angiogenesis. In this study, we show for the first time that in human umbilical vein endothelial cells (HUVECs), 17beta-E2 transiently down-regulates the expression and secretion of a potent negative regulator of angiogenesis, thrombospondin-1 (TSP-1). This inhibitory effect of 17beta-E2 is mediated through nongenomic estrogen receptor (ER)/mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) 1/2 and c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase (SAPK) signaling pathways, because this effect can be abolished by a pure ER antagonist (ICI 182,780) and inhibitors of downstream signaling proteins of MAPK signaling cascades, including MAPK kinase 1/2 and ERK1/2 inhibitor and JNK/SAPK inhibitor. To understand the functional role(s) of TSP-1 during estradiol-induced angiogenesis, we examined the growth and migration of endothelial cells in different experimental environments. Using a recombinant protein, we show that increments of TSP-1 protein concentration in culture medium significantly reduce the migration and proliferation of HUVECs stimulated by 17beta-E2. Together, these studies suggest that TSP-1 can be considered an important negative factor in understanding the increased angiogenesis in response to estrogens.

Topics: Androstadienes; Anthracenes; Antibodies; Butadienes; Cell Division; Cell Movement; Cells, Cultured; Down-Regulation; Endothelial Cells; Estradiol; Estrogen Antagonists; Fulvestrant; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Receptors, Estrogen; RNA, Messenger; Thrombospondin 1; Time Factors; Wortmannin

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.

Topics: Animals; Anthracenes; Butadienes; Cell Differentiation; Cell Line; Chondrocytes; Chondrogenesis; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Imidazoles; Insulin; Mice; Mitogen-Activated Protein Kinases; Nitriles; Pyridines; Signal Transduction

Flavonoids from medicinal plants have been therapeutically administered for cancer therapy. We recently reported that nobiletin (5,6,7,8,3',4'-hexamethoxy flavone) exhibits novel antitumor invasive activities by suppressing the production of pro-matrix metalloproteinases (proMMPs) and augmenting the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) in vivo and in vitro. In the present study, intracellular target molecules associated with the actions of nobiletin against tumor invasion were identified. Nobiletin inhibited the phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2, but not the activity of Ras or the phosphorylation of Raf. Moreover, a MEK1/2 inhibitor, U0126, mimicked nobiletin's ability to decrease the production of proMMPs-1 and 9 in human fibrosarcoma HT-1080 cells stimulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA). In addition, neither the activity of phosphatidylinositol 3-kinase (PI3K) nor the phosphorylation of Akt was influenced by nobiletin. However, nobiletin was found to augment the phosphorylation of c-Jun NH2-terminal kinase (JNK), a downstream signal factor of the PI3K-Akt pathway, in TPA-treated HT-1080 cells. A similar augmentation of JNK phosphorylation was observed on treatment with a PI3K inhibitor, LY-294002. Furthermore, nobiletin enhancement of TIMP-1 production in TPA-stimulated HT-1080 cells was found to be diminished by adding a JNK inhibitor, SP600125. Moreover, protein kinase C (PKC) inhibitor experiments showed that PKCbetaII/epsilon were associated with the nobiletin-mediated augmentation of JNK phosphorylation. Therefore, these results introduce novel evidence that the antitumor effects of nobiletin are finely regulated by the following intracellular mechanisms: (1) the inhibition of MEK1/2 activity is involved in the suppression of MMP expression and (2) the activation of the novel PKCbetaII/epsilon-JNK pathway is associated with the augmentation of TIMP-1 expression.

Topics: Anthracenes; Antineoplastic Agents; Butadienes; Cell Line, Tumor; Collagenases; Enzyme Activation; Enzyme Precursors; Flavones; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Nitriles; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase C; Protein Kinase C beta; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; raf Kinases; ras Proteins; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinase-1

Ectopic calcification of vascular tissue is associated with several cardiovascular pathologies and likely involves active regulation by vascular smooth muscle cells and osteoblast-like vascular cells. This process often occurs in sites with altered mechanical environments, suggesting a role for mechanical stimuli in calcification. In this study, we investigated the effect of mechanical stimulation on the proliferation, osteogenic differentiation, calcification, and mitogen-activated protein kinase (MAPK) signaling in calcifying vascular cells (CVCs), a subpopulation of aortic smooth muscle cells putatively involved in vascular calcification. Application of equibiaxial cyclic strain (7%, 0.25 Hz) to CVCs had no effect on cell proliferation, but accelerated alkaline phosphatase expression and significantly increased mineralization by 3.1-fold over unstrained cells. Fluid motion in the absence of strain also enhanced mineralization, but to a lesser degree. Because MAPK pathways mediate mechanically regulated osteoblast differentiation, we tested whether similar signaling was involved in mineralization by CVCs. In static cultures, pharmacological inhibition of the extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase pathways significantly attenuated mineral production by as much as -94%, compared with uninhibited CVCs. Strikingly, although mechanical stimulation activated each of the MAPK pathways, inhibition of these pathways had no effect on the mechanically induced enhancement of alkaline phosphatase activity or mineralization. These novel data indicate that mechanical signals regulate calcification by CVCs, and although MAPK signaling is critical to CVC osteogenic differentiation and mineralization, it is not involved directly in transduction of mechanical signals to regulate these processes under the conditions utilized in this study.

Topics: Alkaline Phosphatase; Animals; Anthracenes; Butadienes; Calcification, Physiologic; Cattle; Cell Differentiation; Cell Line; Cell Proliferation; DNA; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Imidazoles; JNK Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Nitriles; Osteogenesis; p38 Mitogen-Activated Protein Kinases; Pyridines; Signal Transduction; Stress, Mechanical

Stimulation of alpha1-adrenoceptors induces proliferation of vascular smooth muscle cells (SMCs) and contributes to arterial remodeling. Although activation of NAD(P)H oxidase and generation of reactive oxygen species (ROS) are required, little is known about this pathway. In this study, we examined the hypothesis that epidermal growth factor receptor (EGFR) transactivation and extracellular regulated kinases (ERK) are involved in alpha1-adrenoceptor-mediated SMC growth. Phenylephrine increased protein synthesis in association with a rapid (< or =5 minutes) and sustained (> or =60 minutes) doubling of phosphorylation of EGFR and ERK1/2, but not p38 or JNK in the media of rat aorta maintained in organ culture. Antagonists of EGFR phosphotyrosine activity (AG-1478) and ERK phosphorylation (PD-98059, U-0126) abolished phenylephrine-induced protein synthesis, whereas antagonists of p38 or JNK phosphorylation had no specific effect. A competitive antagonist (P22) for heparin binding EGF-like growth factor (HB-EGF) blocked phenylephrine-induced protein synthesis, as did downregulation of pro-HB-EGF (CRM197). Phenylephrine-induced protein synthesis was inhibited by neutralizing antibody to HB-EGF and absent in HB-EGF-/- SMCs. Inhibitors of metalloproteinases (BiPS, KB-R7785) also blocked adrenergic growth. The neutralizing antibody against HB-EGF had no effect on the two-fold increase in ROS generation induced by phenylephrine (DCF fluorescence), suggesting that stimulation of NAD(P)H oxidase by alpha1-adrenoceptor occupation precedes HB-EGF release. Cell culture studies confirmed and extended these findings. These data suggest that alpha1-adrenoceptor-mediated SMC growth requires ROS-dependent shedding of HB-EGF, transactivation of EGFR, and activation of the MEK1/2-dependent MAP kinase pathway. This trophic pathway may link sympathetic activity to arterial wall growth in adaptive remodeling and hypertrophic disease.

Topics: Adrenergic alpha-1 Receptor Agonists; Animals; Anthracenes; Aorta, Thoracic; Bacterial Proteins; Benzopyrans; Butadienes; Catheterization; Cell Division; Dipeptides; ErbB Receptors; Flavonoids; Glycine; Hydroxamic Acids; Imidazoles; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitriles; Organ Culture Techniques; Phenylephrine; Phosphorylation; Protein Processing, Post-Translational; Pyridines; Quinazolines; Rats; Receptors, Adrenergic, alpha-1; Thrombin; Tyrphostins

To investigate whether activation of mitogen-activated protein kinase (MAPK) in damaged and/or undamaged primary afferents participates in neuropathic pain after partial nerve injury, we examined the phosphorylation of extracellular signal-regulated protein kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) in the L4 and L5 dorsal root ganglion (DRG) in the L5 spinal nerve ligation (SNL) model. We first confirmed, using activating transcription factor 3 and neuropeptide Y immunoreactivity, that virtually all L4 DRG neurons are spared from axotomy in this model. In the injured L5 DRG, the L5 SNL induced the activation of ERK, p38, and JNK in different populations of DRG neurons. In contrast, in the uninjured L4 DRG, the L5 SNL induced only p38 activation in tyrosine kinase A-expressing small- to medium-diameter neurons. Intrathecal ERK, p38, and JNK inhibitor infusions reversed SNL-induced mechanical allodynia, whereas only p38 inhibitor application attenuated SNL-induced thermal hyperalgesia. Furthermore, the L5 dorsal rhizotomy did not prevent SNL-induced thermal hyperalgesia. We therefore hypothesized that p38 activation in the uninjured L4 DRG might be involved in the development of heat hypersensitivity in the L5 SNL model. In fact, the treatment of the p38 inhibitor and also anti-nerve growth factor reduced SNL-induced upregulation of brain-derived neurotrophic factor and transient receptor potential vanilloid type 1 expression in the L4 DRG. Together, our results demonstrate that the L5 SNL induces differential activation of MAPK in injured and uninjured DRG neurons and, furthermore, that MAPK activation in the primary afferents may participate in generating pain hypersensitivity after partial nerve injury.

Topics: Animals; Anthracenes; Brain-Derived Neurotrophic Factor; Butadienes; Enzyme Activation; Ganglia, Spinal; Gene Expression Regulation; Hot Temperature; Hyperalgesia; Imidazoles; Ion Channels; JNK Mitogen-Activated Protein Kinases; Ligation; Lumbar Vertebrae; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons, Afferent; Nitriles; p38 Mitogen-Activated Protein Kinases; Physical Stimulation; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Rhizotomy; Spinal Nerves; Stress, Mechanical; TRPV Cation Channels

Using a metabolic inhibition buffer as an ischemic model, we show here that KR-31378, a cardioselective ATP-sensitive potassium channel opener, protects H9c2 cells from chemical hypoxia (CH)-induced cell death. Our previous study showed that CH downregulated caspase activities, but led to differential activation of mitogen-activated protein kinases (MAPKs) in H9c2 cells. The repression of CH-induced c-jun N-terminal kinase (JNK)/p38 MAPK activation resulted in partial protection against CH- induced cell death, implying JNK/p38 MAPK's causative role in CH-induced cell death. This study furthers that research and examines if KR-31378's protective effect came from modulating MAPK activity and/or caspase activity in H9c2 cells. Although KR-31378 did not restore downregulated caspase-3 activity, it did block the activation of JNK and p38 MAPK in a dose-dependent manner. Extracellular signal-regulated kinase activity was not recovered by KR-31378 treatment. CH-induced reactive oxygen species (ROS) generation was suppressed by KR-31378. Thus our results indicate that the cardioprotective effect of KR-31378 in CH is due, at least in part, to the differential inhibition of MAPKs.

Topics: Animals; Anthracenes; Butadienes; Cell Death; Cell Hypoxia; Cell Line; Enzyme Activation; Guanidines; Imidazoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Molecular Structure; Myocardium; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyrans; Pyridines; Reactive Oxygen Species

Interleukin (IL)-8 serves as a major chemoattractant for neutrophils and has also been proposed to affect cancer progression. In the present study, we show that IGF-I stimulates IL-8 mRNA expression and IL-8 secretion in the leukemic cell line HL-60. Stimulation of IL-8 expression was completely attenuated by two inhibitors of mitogen-activated protein kinase (MAPK) kinase (MEK), which phosphorylates the MAPKs extracellular-regulated kinase (ERK)1 and ERK2, and by the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. In contrast, inhibitors of p38 MAPK and phosphatidylinositol-3 kinase (PI3K) did not abrogate the effect of IGF-I. We also show that IGF-I stimulates the activation of ERK1 and ERK2, but we could not detect any effect of IGF-I on the phosphorylation of p38, JNK(p46) or JNK(p54). Collectively, our results suggest that basal JNK activity and activation of the MEK-ERK pathway are required for upregulation of IL-8 by IGF-I in HL-60 cells.

Topics: Androstadienes; Anthracenes; Apoptosis; Butadienes; Cell Survival; Chromones; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Hydrogen Peroxide; Imidazoles; Insulin-Like Growth Factor I; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Kinetics; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Oxazoles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Receptor, IGF Type 1; RNA, Messenger; Wortmannin

p8 is a stress-induced protein with multiple functions and biochemically related to the architectural factor HMG-I/Y. We analyzed the expression and function of p8 in pancreatic cancer-derived cells.. Expression of p8 was silenced in the human pancreatic cancer cell lines Panc-1 and BxPc-3 by infection with a retrovirus expressing p8 RNA in the antisense orientation. Cell growth was measured in control and p8-silenced cells. Influence on p8 expression of the induction of intracellular pathways promoting cellular growth or growth arrest was monitored.. p8-silenced cells grew more rapidly than control cells transfected with the empty retrovirus. Activation of the Ras-->Raf-->MEK-->ERK and JNK intracellular pathways down-regulated p8 expression. In addition, the MEK1/2 inhibitor U0126 and the JNK inhibitor SP600125 up-regulates expression of p8. Conversely, p38 or TGFbeta-1 induced p8 expression whereas the specific p38 inhibitor SB203580 down-regulated p8 expression. Finally, TGFbeta-1 induction was in part mediated through p38.. p8 inhibits the growth of human pancreatic cancer cells. p8 expression is induced through pathways involved in growth inhibition and repressed by factors that promote cell growth. These results suggest that p8 belongs to a pathway regulating the growth of pancreatic cancer cells.

Topics: Anthracenes; Basic Helix-Loop-Helix Transcription Factors; Butadienes; Cell Line, Tumor; Cell Proliferation; Culture Media; Culture Media, Serum-Free; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Vectors; Growth Inhibitors; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nitriles; Pancreatic Neoplasms; Proto-Oncogene Proteins c-raf; Pyridines; ras Proteins; Retroviridae; RNA, Antisense; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1