u-0126 and Inflammation

Women's fecundity is known to decrease with the increase in chronologic age. Several biomarkers of the ovarian reserve, including follicle stimulating hormone (FSH), anti Müllerian hormone (AMH), have been proposed as possible predictors for the response to controlled ovarian stimulation (COS). Although there are assumptions indicating that the relationship between age and ovarian reserve is highly variable and the potential different validity of ovarian reserve markers in women in different age groups remains to be demonstrated. The purpose of our study was evaluating FSH and AMH as potential predictors of response to controlled ovarian stimulation and prediction of intracytoplasmic sperm injection (ICSI) outcome according to age. This prospective study has been carried out on 218 women having ICSI cycles. Cases were divided into two groups, group 1 (. We found that below the age of 35 years, the chances of pregnancy are more correlated to FSH levels, while above the age of 35 years, AMH was a more relevant test.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Basigin; beta-Transducin Repeat-Containing Proteins; Brain Neoplasms; Butadienes; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diabetic Cardiomyopathies; Diet, High-Fat; Dietary Supplements; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Endoribonucleases; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; NF-E2-Related Factor 2; Nitriles; Phosphorylation; Rats; Signal Transduction; Sumoylation; Temozolomide; Unfolded Protein Response; X-Box Binding Protein 1; Xenograft Model Antitumor Assays

Infertility is a common disease among women of childbearing age and seriously endangers the reproductive health of human beings.. We aimed to study the active effect and mechanism of betulonic acid (BTA) on tubal inflammatory infertility.. An inflammatory model was established in isolated rat oviduct epithelial cells. Immunofluorescence of cytokeratin 18 was performed in cells. The therapeutic effect of BTA on cells was observed. Subsequently, we added JAK/STAT inhibitor AG490 and MAPK inhibitor U0126 and measured the levels of inflammatory factors via enzyme-linked immunosorbent assay and qRT-PCR. CCK-8 assay was applied to test cell proliferation, whereas flow cytometry was used to measure apoptosis. The levels of TLR4, IκBα, JAK1, JAK2, JAK3, Tyk2, STAT3, p38, ERK and the phosphorylation of p65 were determined by Western blotting.. Betulonic acid inhibited the activation of TLR4 and NF-κB signalling pathways, and significantly downregulated IL-1β, IL-6, and TNF-α, with high doses being the most effective. Furthermore, high-dose BTA promoted the proliferation of oviduct epithelial cells and inhibited apoptosis. In addition, BTA inhibited the activation of JAK/STAT signalling pathway to perform effectively in oviduct epithelial cells inflammation. The addition of AG490 led to the inhibition of the JAK/STAT signalling pathway. BTA also inhibited the activation of MAPK signalling pathway in oviduct epithelial cells inflammation. Under U0126 treatment, the inhibition of proteins in MAPK pathway by BTA was weakened.. Therefore, BTA inhibited the TLR, JAK/STAT and MAPK signalling pathways.. Our study provided a new therapeutic strategy for infertility caused by oviduct inflammation.

Topics: Animals; Epithelial Cells; Female; Humans; Inflammation; NF-kappa B; Oviducts; Rats; Toll-Like Receptor 4

This study focused on the relationship between extracellular-regulated kinase (ERK) and obesity-induced increases in neuropathic pain. We fed rats a high-fat diet to establish the obesity model, and rats were given surgery to establish the chronic compression of the dorsal root ganglia (CCD) model. U0126 was applied to inhibit ERK, and metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) was applied to cause AMP-activated protein kinase (AMPK) activation. Paw withdrawal mechanical threshold (PWMT) were calculated to indicate the level of neuropathic pain. The data indicated that compared with normal CCD rats, the PWMT of obese CCD rats were decreased, accompanied with an increase of ERK phosphorylation, NAD(P)H oxidase 4 (NOX4) protein expression, oxidative stress and inflammatory level in the L4 to L5 spinal cord and dorsal root ganglia (DRG). Administration of U0126 could partially elevate the PWMT and reduce the protein expression of NOX4 and the above pathological changes in obese CCD rats.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Apoptosis; Butadienes; Diet, High-Fat; Disease Models, Animal; Enzyme Inhibitors; Ganglia, Spinal; Hypoglycemic Agents; Inflammation; Male; MAP Kinase Signaling System; Metformin; NADPH Oxidase 4; Neuralgia; Nitriles; Obesity; Oxidative Stress; Pain Threshold; Phosphorylation; Rats, Wistar; Ribonucleotides; Spinal Cord

Obstructive sleep apnea syndrome (OSAS) is a common and complex disorder that is associated with liver injury. Moreover, previous studies have revealed that chronic intermittent hypoxia (CIH) is associated with the development of non‑alcoholic fatty liver disease and hepatic fibrosis. However, the underlying molecular mechanisms remain largely unknown. The present study aimed to investigate whether chronic intermittent hypoxia induced hepatic fibrosis, in addition to determining its underlying mechanisms, in CIH model rats using immunohistochemistry, western blotting and reverse transcription‑quantitative PCR. The present results suggested that CIH caused hepatic fibrosis and increased the expression levels of interleukin (IL)‑1β, IL‑8, monocyte chemotactic‑1, tumor necrosis factor‑α, intercellular adhesion molecule‑1 and vascular cell adhesion molecule‑1 in the liver; these conditions could be reversed by Toll‑like receptor 4 (TLR4) short hairpin RNA lentivirus treatment. Moreover, immunohistochemistry and western blotting results indicated that TLR4 and NF‑κB expression levels were significantly increased in the CIH and CIH‑TLR4 empty vector lentivirus group. However, protein expression levels of TLR4, NF‑κB, inhibitor of NF‑κB and phosphorylated‑mitogen‑activated protein kinase (MAPK)‑1 in the hypoxia/reoxygenation group were significantly higher compared with the control group (P<0.05), and these results were reversed by the MAPK inhibitor U0126 in vitro. Collectively, the present preliminary results suggested that inflammation and the TLR4/NF‑κB/MAPK signaling pathway may be involved in CIH‑induced liver fibrosis.

Topics: Animals; Butadienes; Cell Line; Disease Models, Animal; Enzyme Inhibitors; Gene Silencing; Hepatic Stellate Cells; Hypoxia; Inflammation; Liver Cirrhosis; Male; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Rats; Rats, Sprague-Dawley; Signal Transduction; Sleep Apnea, Obstructive; Toll-Like Receptor 4

Previous research demonstrated that IL-10 was up-regulated in Chlamydia trachomatis-infected cells and that exogenous IL-10 was able to inhibit the secretion of pro-inflammatory cytokines by infected cells. However, the mechanisms are not well understood. The aim of this study was to investigate the mechanisms for up-regulation of IL-10 and inhibition of pro-inflammatory cytokine secretion in C. trachomatis-stimulated peripheral blood mononuclear cells (PBMCs).. Human PBMCs were isolated from the blood of healthy human donors by standard Ficoll-Hypaque density gradient centrifugation. Cells were exposed to C. trachomatis in the presence or absence of MEK inhibitor U0126, the p38 inhibitor SB203580, the STAT3 inhibitor Ruxolitinib or anti-human IL-10 antibody. Cytokines were measured from culture supernatants using ELISA kits. Cells were harvested for real-time quantitative PCR to determine IL-10 mRNA levels and for Western blot assay to detect the expression of ERK1/2, p-ERK1/2, p38, p-p38, STAT3 and p-STAT3.. Both mRNA and protein levels of IL-10 were up-regulated in stimulated cells, and the production of IL-10 was reduced when cells were treated with U0126 or SB203580. The expression of cytokines IL-6, IL-8 and TNF-α was enhanced in stimulated cells treated with anti-human IL-10 antibody. Moreover, neutralization of IL-10 resulted in a significant decrease of phosphorylated STAT3 in stimulated cells. Ruxolitinib caused a significant increase in the production of IL-6, IL-8 and TNF-α in stimulated cells.. IL-10 is up-regulated in an ERK- and p38-dependent fashion in stimulated human PBMCs. IL-10 inhibits the production of pro-inflammatory cytokines by activating the JAK/STAT signalling pathway.

Topics: Butadienes; Chlamydia trachomatis; Cytokines; Enzyme Inhibitors; Humans; Imidazoles; Inflammation; Interleukin-10; Interleukin-6; Interleukin-8; Leukocytes, Mononuclear; Mitogen-Activated Protein Kinase 1; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyrazoles; Pyridines; Pyrimidines; Real-Time Polymerase Chain Reaction; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha; Up-Regulation

Immunoglobulin A nephropathy (IgAN) is one of the most common glomerular diseases worldwide. Various studies have identified a host of microRNAs (miRNAs) abnormally expressed in IgAN and might affect the pathogenesis and progression of IgAN. However, miR-200bc/429 cluster in the pathopoiesis of IgAN remains poorly understood. For this study, we found that miR-200bc/429 cluster is downregulated in IgAN tissues and IgAN podocytes and HK2 cells compared with their matched controls respectively. In addition, overexpression of miR-200bc/429 cluster in IgAN podocytes and HK2 cells could attenuate the release of inflammatory cytokines MCP-1, IL-6 and RANTES. Moreover, the 3' untranslated region (UTR) of TNF-like weak inducer of apoptosis (TWEAK) was identified to be a direct target of miR-200bc/429 cluster. Furthermore, our results showed that miR-200bc/429 cluster can inhibit TWEAK mediated NF-κB pathway activation in IgAN. Overall, our findings revealed that miR-200bc/429 cluster alleviates inflammation in IgAN through TWEAK/Fn14 system and might serve as a biomarker as well as a promising therapeutic target for IgAN.

Topics: Aminosalicylic Acids; Animals; Apoptosis; Benzenesulfonates; Butadienes; Cell Line; Cytokine TWEAK; Glomerulonephritis, IGA; Glycation End Products, Advanced; Inflammation; Interleukin-6; MAP Kinase Signaling System; Mice; MicroRNAs; Nitriles; Osteocytes; p38 Mitogen-Activated Protein Kinases; STAT3 Transcription Factor; Vascular Endothelial Growth Factor A

Atherosclerosis is a chronic inflammatory disease, which is thought to be one of the most common causes of death globally. The functions of macrophage in the development of atherosclerosis inflammation still get more attention. Although lipopolysaccharide (LPS) can trigger inflammation in atherosclerosis, how LPS promotes atherogenesis through acting on macrophage is not very clear. Here, we study the role of adipophilin in LPS-induced inflammation. After RAW264.7 cells were treated with LPS of different concentrations, the protein level of adipophilin was increased dose-dependently, and cells treated with LPS for various time were observed the highest levels of TNF-α, MCP-1, and IL-6 at 12 h. In addition, inhibited extracellular signal-regulated kinase (ERK)-1/2 presented lower levels of adipophilin, peroxisome proliferator-activated receptor gamma (PPARγ), TNF-α, MCP-1, as well as IL-6. But inhibited PPARγ, the levels of adipophilin, TNF-α, MCP-1, and IL-6 were significantly augmented. Moreover, after silence adipophilin, the ERK1/2 activity and protein level of PPARγ were not influenced, whereas the levels of TNF-α, MCP-1, and IL-6 were significantly reduced. LPS can promote the expression of adipophilin through ERK1/2-PPARγ pathway, whereby it enhances the secretion levels of TNF-α, MCP-1, and IL-6.

Topics: Animals; Atherosclerosis; Butadienes; Cytokines; Extracellular Signal-Regulated MAP Kinases; Inflammation; Lipopolysaccharides; Macrophages; MAP Kinase Signaling System; Mice; Nitriles; Perilipin-2; PPAR gamma; Protein Kinase Inhibitors; RAW 264.7 Cells; RNA, Small Interfering; Signal Transduction

Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by pathological osteogenesis and inflammation. However, the pathogenesis of AS and the pathological relationship between osteogenesis and inflammation in this disease remain largely unknown. Mesenchymal stem cells (MSCs) are multipotent progenitor cells capable of osteogenic differentiation and immunoregulation. Recently, we demonstrated that MSCs from AS patients (ASMSCs) have a greater potential for osteogenic differentiation than MSCs from healthy donors (HDMSCs), which therefore seems to be a component of pathological osteogenesis in AS. Previous studies have indicated that the immunoregulatory abilities of MSCs change following differentiation. However, the subsequent effects of ASMSCs during abnormal osteogenic differentiation are unclear. Here, we further demonstrated that ASMSCs secreted more monocyte chemoattractant protein 1 (MCP1) than HDMSCs during osteogenic differentiation. This enhanced MCP1 secretion augmented monocyte migration, increased classical macrophage polarization, and enhanced TNF-α secretion. Inhibiting MCP1 secretion from osteogenic differentiated ASMSCs using lentiviruses encoding short hairpin RNAs ameliorated these dysfunctions. Blocking the ERK1/2 pathway in ASMSCs with U0126 corrected the abnormal osteogenic differentiation, inhibited MCP1 overexpression, and prevented subsequent monocyte dysfunction. Finally, MCP1 expression was up-regulated during osteogenic differentiation in ASMSCs in vivo and was locally augmented in osteoblasts at ossification sites in AS patients. In summary, our study determined that MCP1 overexpression during abnormal osteogenic differentiation of ASMSCs triggers monocyte dysfunctions. We propose the novel hypothesis that pathological osteogenesis can lead to inflammation in AS. This hypothesis may contribute to reveal the precise pathological relationship between osteogenesis and inflammation in the field of osteoimmunology.. ASMSCs secreted more MCP1 during abnormal osteogenic differentiation. MCP1 overexpression leads to monocyte dysfunctions. Pathological osteogenesis can lead to inflammation in AS.

Topics: Adult; Butadienes; Cell Differentiation; Cells, Cultured; Chemokine CCL2; Enzyme Inhibitors; Female; Humans; Inflammation; Macrophages; Male; MAP Kinase Signaling System; Mesenchymal Stem Cells; Monocytes; Nitriles; Osteoblasts; Osteogenesis; RNA, Small Interfering; Spondylitis, Ankylosing; Tumor Necrosis Factor-alpha

Neutrophil elastase plays a crucial role in arthritis. Here, its potential in triggering joint inflammation and pain was assessed, and whether these effects were mediated by proteinase-activated receptor-2 (PAR2).. Neutrophil elastase (5 μg) was injected into the knee joints of mice and changes in blood perfusion, leukocyte kinetics and paw withdrawal threshold were assessed. Similar experiments were performed in animals pretreated with the neutrophil elastase inhibitor sivelestat, the PAR2 antagonist GB83, the p44/42 MAPK inhibitor U0126 and in PAR2 receptor knockout (KO) mice. Neutrophil elastase activity was also evaluated in arthritic joints by fluorescent imaging and sivelestat was assessed for anti-inflammatory and analgesic properties.. Intra-articular injection of neutrophil elastase caused an increase in blood perfusion, leukocyte kinetics and a decrease in paw withdrawal threshold. Sivelestat treatment suppressed this effect. The PAR2 antagonist GB83 reversed neutrophil elastase-induced synovitis and pain and these responses were also attenuated in PAR2 KO mice. The MAPK inhibitor U0126 also blocked neutrophil elastase-induced inflammation and pain. Active neutrophil elastase was increased in acutely inflamed knees as shown by an activatable fluorescent probe. Sivelestat appeared to reduce neutrophil elastase activity, but had only a moderate anti-inflammatory effect in this model.. Neutrophil elastase induced acute inflammation and pain in knee joints of mice. These changes are PAR2-dependent and appear to involve activation of a p44/42 MAPK pathway. Blocking neutrophil elastase, PAR2 and p44/42 MAPK activity can reduce inflammation and pain, suggesting their utility as therapeutic targets.

Topics: Animals; Butadienes; Inflammation; Knee Joint; Leukocyte Elastase; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitriles; Pain; Receptor, PAR-2

Infection with enterohemorrhagic Escherichia coli (EHEC) can result in severe disease, including hemorrhagic colitis and the hemolytic uremic syndrome. Shiga toxins (Stx) are the key EHEC virulence determinant contributing to severe disease. Despite inhibiting protein synthesis, Shiga toxins paradoxically induce the expression of proinflammatory cytokines from various cell types in vitro, including intestinal epithelial cells (IECs). This effect is mediated in large part by the ribotoxic stress response (RSR). The Shiga toxin-induced RSR is known to involve the activation of the stress-activated protein kinases (SAPKs) p38 and JNK. In some cell types, Stx also can induce the classical mitogen-activated protein kinases (MAPKs) or ERK1/2, but the mechanism(s) by which this activation occurs is unknown. In this study, we investigated the mechanism by which Stx activates ERK1/2s in IECs and the contribution of ERK1/2 activation to interleukin-8 (IL-8) expression. We demonstrate that Stx1 activates ERK1/2 in a biphasic manner: the first phase occurs in response to StxB1 subunit, while the second phase requires StxA1 subunit activity. We show that the A subunit-dependent ERK1/2 activation is mediated through ZAK-dependent signaling, and inhibition of ERK1/2 activation via the MEK1/2 inhibitors U0126 and PD98059 results in decreased Stx1-mediated IL-8 mRNA. Finally, we demonstrate that ERK1/2 are activated in vivo in the colon of Stx2-intoxicated infant rabbits, a model in which Stx2 induces a primarily neutrophilic inflammatory response. Together, our data support a role for ERK1/2 activation in the development of Stx-mediated intestinal inflammation.

Topics: Animals; Butadienes; Cell Line; Enterohemorrhagic Escherichia coli; Enzyme Activation; Epithelial Cells; Escherichia coli Infections; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); HEK293 Cells; Hemolytic-Uremic Syndrome; Humans; Inflammation; Interleukin-8; Intestinal Mucosa; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinases; Nitriles; p38 Mitogen-Activated Protein Kinases; Protein Kinases; Rabbits; RNA Interference; RNA, Small Interfering; Shiga Toxin 1; Shiga Toxin 2

We previously reported the involvement of serotonin (5-HT) metabolism in cigarette smoke-induced oxidative stress in rat lung in vivo. Here, we report cigarette smoke as a source of serotonin (5-HT) to the airways and aim at investigating the effects of 5-HT on oxidative stress and inflammation in human bronchial epithelial cells (BEAS-2B). A 5-HT analog was identified to be present in aqueous phase cigarette smoke using the LC-MS/MS approach, which was later confirmed by a 5-HT enzyme-linked immune assay (EIA). Furthermore, exposure to 5-HT caused a time-dependent elevation of intracellular ROS level, which was blocked in the presence of apocynin (a NOX inhibitor). In support, the immunoblot analysis indicated that there was an increase in the expression of NOX2 time-dependently. 5-HT-induced elevation of IL-8 at both mRNA and protein levels was observed, which was inhibited by TEMPOL (a free radical scavenger), and inhibitors for p38 MAPK (SB203580) and ERK (U0126), in line with the time-dependent phosphorylation of p38 MAPK and ERK. In conclusion, our findings suggest that 5-HT presented in bronchial epithelium of smokers may be involved in cigarette smoke-induced oxidative stress and inflammation via activation of p38 MAPK and ERK pathway after the formation of free radicals.

Topics: Alveolar Epithelial Cells; Animals; Antioxidants; Butadienes; Cyclic N-Oxides; Free Radicals; Humans; Imidazoles; Inflammation; MAP Kinase Signaling System; Nitriles; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Reactive Oxygen Species; Serotonin; Smoking; Spin Labels; Tandem Mass Spectrometry

This paper aims to observe the changes of the inflammatory cytokines in microglial BV2 cells stimulated by apelin, and investigate the mechanism of inflammatory cytokines secretion after apelin stimulation. Immunofluorescence and quantitative real-time PCR were performed to observe expression of TNF-α, IL-1β, IL-10, MIP-1α, and MCP-1 in BV2 cells. Western blot was used to investigate the expression of phosphorylation PI-3K/Akt and phosphorylation Erk signaling pathways in BV2 cells after stimulation by apelin. Furthermore, PI-3K/Akt inhibitor (LY294402) and Erk inhibitor (U0126) were used as antagonists to detect the secretion mechanisms of cytokines in BV2 cells stimulated by apelin. Exogenous recombinant apelin activated the expression of TNF-α, IL-1β, MCP-1 and MIP-1α in BV2 cells by the detection of fluorescence expression and mRNA. Apelin also unregulated the protein expression of p-PI-3K/Akt and p-Erk in BV2 cells induced by apelin. LY294002 and U0126 inhibited activation of p-PI-3K/Akt and p-Erk expression by Western blot and attenuated the expression of inflammation factors in BV2 cells by fluorescence staining. This study demonstrates that apelin is a potential activator of inflammation factors through the PI3K/Akt and Erk signaling pathway and is potential therapeutically relevant to inflammatory responses of microglia cells.

Topics: Adipokines; Animals; Apelin; Apelin Receptors; Butadienes; Chemokine CCL2; Chemokine CCL3; Chromones; Enzyme Inhibitors; Gene Expression Regulation; Inflammation; Intercellular Signaling Peptides and Proteins; Interleukin-10; Interleukin-1beta; Mice; Microglia; Microscopy, Fluorescence; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Real-Time Polymerase Chain Reaction; Receptors, G-Protein-Coupled; Recombinant Proteins; Signal Transduction; Tumor Necrosis Factor-alpha

Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3'-untranslated region (3'-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3'-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3'-UTR. Transcript destabilization by TTP was nullified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation.

Topics: Animals; AU Rich Elements; Butadienes; Gene Expression Regulation; HEK293 Cells; Humans; Inflammation; Interleukin-22; Interleukins; Jurkat Cells; MAP Kinase Kinase 1; Mice; Nitriles; Primary Cell Culture; RNA, Messenger; STAT3 Transcription Factor; T-Lymphocytes; Tristetraprolin

Epidermal growth factor (EGF) and their receptor (EGFR) play an important role in the development of cancer proliferation, and metastasis, although the mechanism remains unclear. The present study aimed at investigating the role of EGF-EGFR signalling pathway in the development of human hepatocellular carcinoma (HCC) inflammatory environment. Gene profiles of inflammatory cytokines from HCC were measured. Cell bio-behaviours of HCC with low or high metastasis were detected by the live cell monitoring system. Cell proliferation was measured by CCK8. The protein level of CXCL5 and CXCL8 was measured by ELISA. The phosphorylation of PI3K, ERK, MAPK was measured by western blot. EGF significantly induced cell proliferation in HepG2 cells, but not in HCCLM3 cells. EGF prompted the cell movement in both HepG2 and HCCLM3 and regulated the production of CXCL5 and CXCL8 from HCC, which were inhibited by EGFR inhibitor, Erk inhibitor (U0126), or PI3K inhibitors (BEZ-235 and SHBM1009). HCC proliferation, metastasis and production of inflammatory cytokines were regulated via EGF-EGFR signal pathways. CXCL5 could interact with CXCL8, possibly by CXCR2 or the cross-talk between CXCR2 and EGFR. EGF-EGFR signaling pathway can be the potential target of therapies for HCC.

Topics: Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CXCL5; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Inflammation; Interleukin-8; Liver Neoplasms; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Quinolines; Receptor Cross-Talk; Receptors, Interleukin-8B; Signal Transduction; Tumor Microenvironment

Skeletal muscle insulin resistance in type 2 diabetes is associated with a shift from oxidative to glycolytic metabolism in myofibers. However, whether this metabolic switch is detrimental or adaptive for metabolic homeostasis has not been resolved. We recently demonstrated that the Baf60c/Deptor pathway promotes glycolytic metabolism in the muscle and protects mice from diet-induced insulin resistance. However, the nature of the signals that impinge on this pathway and the role of Baf60c in glucose homeostasis in the severe insulin-resistant state remain unknown. Here we show that expression of Baf60c and Deptor was downregulated in skeletal muscle in obesity, accompanied by extracellular signal-related kinase (ERK) activation. In cultured myotubes, inhibition of ERK, but not Jun NH2-terminal kinase and IκB kinase, blocked the downregulation of Baf60c and Deptor by the proinflammatory cytokine tumor necrosis factor-α. Treatment of obese mice with the ERK inhibitor U0126 rescued Baf60c and Deptor expression in skeletal muscle and lowered blood glucose. Transgenic rescue of Baf60c in skeletal muscle restored Deptor expression and Akt phosphorylation and ameliorated insulin resistance in ob/ob mice. This study identifies the Baf60c/Deptor pathway as a target of proinflammatory signaling in skeletal muscle that may link meta-inflammation to skeletal myofiber metabolism and insulin resistance.

Topics: Animals; Butadienes; Chromosomal Proteins, Non-Histone; Down-Regulation; Enzyme Inhibitors; Glucose; Glucose Tolerance Test; Homeostasis; I-kappa B Kinase; Inflammation; Insulin; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Mice; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Nitriles; Obesity; Signal Transduction; Tumor Necrosis Factor-alpha

Cachexia is a relevant comorbid condition of chronic diseases including cancer. Inflammation, oxidative stress, autophagy, ubiquitin-proteasome system, nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) are involved in the pathophysiology of cancer cachexia. Currently available treatment is limited and data demonstrating effectiveness in in vivo models are lacking. Our objectives were to explore in respiratory and limb muscles of lung cancer (LC) cachectic mice whether proteasome, NF-κB, and MAPK inhibitors improve muscle mass and function loss through several molecular mechanisms. Body and muscle weights, limb muscle force, protein degradation and the ubiquitin-proteasome system, signaling pathways, oxidative stress and inflammation, autophagy, contractile and functional proteins, myostatin and myogenin, and muscle structure were evaluated in the diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing cachectic mice (BALB/c), with and without concomitant treatment with NF-κB (sulfasalazine), MAPK (U0126), and proteasome (bortezomib) inhibitors. Compared to control animals, in both respiratory and limb muscles of LC cachectic mice: muscle proteolysis, ubiquitinated proteins, autophagy, myostatin, protein oxidation, FoxO-1, NF-κB and MAPK signaling pathways, and muscle abnormalities were increased, while myosin, creatine kinase, myogenin, and slow- and fast-twitch muscle fiber size were decreased. Pharmacological inhibition of NF-κB and MAPK, but not the proteasome system, induced in cancer cachectic animals, a substantial restoration of muscle mass and force through a decrease in muscle protein oxidation and catabolism, myostatin, and autophagy, together with a greater content of myogenin, and contractile and functional proteins. Attenuation of MAPK and NF-κB signaling pathway effects on muscles is beneficial in cancer-induced cachexia.

Topics: Animals; Autophagy; Boronic Acids; Bortezomib; Butadienes; Cachexia; Cell Line, Tumor; Inflammation; Lung Neoplasms; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle Proteins; Muscle Weakness; Muscle, Skeletal; NF-kappa B; Nitriles; Oxidation-Reduction; Proteolysis; Pyrazines; Signal Transduction; Sulfasalazine; Tyrosine; Weight Gain

Canonical neutrophil antimicrobial effector mechanisms, such as degranulation, production of reactive oxygen species, and release of neutrophil extracellular traps (NETs), can result in severe pathology. Activation of neutrophils through immune complexes (ICs) plays a central role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report that immobilized ICs (iICs), which are hallmarks of several autoimmune diseases, induce the release of NETs from primary human neutrophils. The iIC-induced NET formation was found to require production of reactive oxygen species by NADPH oxidase and myeloperoxidase and to be mediated by FcγRIIIb. Blocking of the β2 integrin macrophage-1 Ag but not lymphocyte function-associated Ag-1 abolished iIC-induced NET formation. This suggests that FcγRIIIb signals in association with macrophage-1 Ag. As intracellular signaling pathways involved in iIC-induced NET formation we identified the tyrosine kinase Src/Syk pathway, which downstream regulates the PI3K/Akt, p38 MAPK, and ERK1/2 pathways. To our knowledge, the present study shows for the first time that iICs induce NET formation. Thus, we conclude that NETs contribute to pathology in autoimmune inflammatory disorders associated with surface-bound ICs.

Topics: Aminopyrine; Antigen-Antibody Complex; Antioxidants; Ascorbic Acid; Autoimmune Diseases; Butadienes; CD11a Antigen; CD18 Antigens; Cell Degranulation; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; GPI-Linked Proteins; Humans; Imidazoles; Inflammation; Intracellular Signaling Peptides and Proteins; Lymphocyte Function-Associated Antigen-1; Macrophage-1 Antigen; Mesalamine; Neutrophil Activation; Neutrophils; Nitriles; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Reactive Oxygen Species; Receptors, IgG; src-Family Kinases; Syk Kinase

Tumor cells require increased adenosine triphosphate (ATP) to support anabolism and proliferation. The precise mechanisms regulating this process in tumor cells are unknown. Here, we show that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), are required for optimal mitochondrial function within tumors. We found that RAGE is present in the mitochondria of cultured tumor cells as well as primary tumors. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. These findings link, for the first time, the HMGB1-RAGE pathway with changes in bioenergetics. Moreover, our observations provide a novel mechanism within the tumor microenvironment by which necrosis and inflammation promote tumor progression.

Topics: Adenosine Triphosphate; Animals; Butadienes; CD24 Antigen; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cycloheximide; Electron Transport Complex I; Energy Metabolism; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; HMGB1 Protein; Humans; Inflammation; MAP Kinase Kinase 2; Mice; Mitochondria; NF-kappa B; Nitriles; Pancreatic Neoplasms; Phosphorylation; Protein Binding; Protein Synthesis Inhibitors; Receptor for Advanced Glycation End Products; RNA Interference; RNA, Small Interfering; Rotenone; Signal Transduction; Toll-Like Receptor 2; Toll-Like Receptor 4; Tumor Microenvironment; Uncoupling Agents

The C/EBP β is a basic leucine zipper transcription factor that regulates a variety of biological processes, including metabolism, cell proliferation and differentiation, and immune response. Recent findings show that C/EBP β-induced inflammatory responses mediate kainic acid-triggered excitotoxic brain injury. In this article, we show that protein kinase C ζ enhances K-ras expression and subsequently activates the Raf/MEK/ERK1/2 pathway in the hippocampus of domoic acid (DA)-treated mice, which promotes C/EBP β expression and induces inflammatory responses. Elevated production of TNF-α impairs mitochondrial function and increases the levels of reactive oxygen species by IκB kinase β/NF-κB signaling. The aforementioned inflammation and oxidative stress lead to memory deficits in DA-treated mice. However, troxerutin inhibits cyclin-dependent kinase 1 expression, enhances type 1 protein phosphatase α dephosphorylation, and abolishes MEK/ERK1/2/C/EBP β activation, which subsequently reverses the memory impairment observed in the DA-treated mice. Thus, troxerutin is recommended as a potential candidate for the prevention and therapeutic treatment of cognitive deficits resulting from excitotoxic brain damage and other brain disorders.

Topics: Animals; Butadienes; CCAAT-Enhancer-Binding Protein-beta; CDC2 Protein Kinase; Gene Knockdown Techniques; Genes, ras; Hippocampus; Hydroxyethylrutoside; Inflammation; Inflammation Mediators; Kainic Acid; Male; Memory Disorders; Mice; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NADPH Oxidases; Nitriles; Oxidative Stress; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Reactive Oxygen Species; Signal Transduction

Monocytes and macrophages are important innate immune cells equipped with danger-sensing receptors, including complement and Toll-like receptors. Complement protein C5a, acting via C5aR, is shown in this study to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. Whereas C5a enhanced secretion of LPS-induced IL-6 and TNF from primary human monocytes, C5a inhibited these responses while increasing IL-10 secretion in donor-matched human monocyte-derived macrophages differentiated by GM-CSF or M-CSF. Gαi/c-Raf/MEK/ERK signaling induced by C5a was amplified in macrophages but not in monocytes by LPS. Accordingly, the Gαi inhibitor pertussis toxin and MEK inhibitor U0126 blocked C5a inhibition of LPS-induced IL-6 and TNF production from macrophages. This synergy was independent of IL-10, PI3K, p38, JNK, and the differentiating agent. Furthermore, C5a did not inhibit IL-6 production from macrophages induced by other TLR agonists that are selective for Toll/IL-1R domain-containing adapter inducing IFN-β (polyinosinic-polycytidylic acid) or MyD88 (imiquimod), demonstrating selectivity for C5a regulation of LPS responses. Finally, suppression of proinflammatory cytokines IL-6 and TNF in macrophages did not compromise antimicrobial activity; instead, C5a enhanced clearance of the Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium from macrophages. C5aR is thus a regulatory switch that modulates TLR4 signaling via the Gαi/c-Raf/MEK/ERK signaling axis in human macrophages but not monocytes. The differential effects of C5a are consistent with amplifying monocyte proinflammatory responses to systemic danger signals, but attenuating macrophage cytokine responses (without compromising microbicidal activity), thereby restraining inflammatory responses to localized infections.

Topics: Aminoquinolines; Butadienes; Cells, Cultured; Complement C5a; Extracellular Signal-Regulated MAP Kinases; Granulocyte-Macrophage Colony-Stimulating Factor; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Imiquimod; Inflammation; Interleukin-10; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Macrophage Colony-Stimulating Factor; Macrophages; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Monocytes; Nitriles; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Poly I-C; Proto-Oncogene Proteins c-raf; Receptor, Anaphylatoxin C5a; Receptors, Complement; Salmonella typhimurium; Toll-Like Receptor 4; Tumor Necrosis Factors

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

Radiation-induced normal brain injury is associated with acute and/or chronic inflammatory responses, and has been a major concern in radiotherapy. Recent studies suggest that microglial activation is a potential contributor to chronic inflammatory responses following irradiation; however, the molecular mechanism underlying the response of microglia to radiation is poorly understood. c-Jun, a component of AP-1 transcription factors, potentially regulates neural cell death and neuroinflammation. We observed a rapid increase in phosphorylation of N-terminal c-Jun (on serine 63 and 73) and MAPK kinases ERK1/2, but not JNKs, in irradiated murine microglial BV2 cells. Radiation-induced c-Jun phosphorylation is dependent on the canonical MEK-ERK signaling pathway and required for both ERK1 and ERK2 function. ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases. Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway. Radiation stimulates c-Jun transcriptional activity and upregulates c-Jun-regulated proinflammatory genes, such as tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2. Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes. Overall, our study reveals that the MEK-ERK1/2 signaling pathway, but not the JNK pathway, contributes to the c-Jun-dependent microglial inflammatory response following irradiation.

Topics: Animals; Base Sequence; Brain Injuries; Butadienes; Cell Line; DNA Primers; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Inflammation; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Microglia; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Radiation Injuries, Experimental; Reactive Oxygen Species; Recombinant Fusion Proteins; Transcription, Genetic

Inflammation in Parkinson's disease is closely associated with disease pathogenesis. Mutations in Omi, which encodes the protease Omi, are linked to neurodegeneration and Parkinson's disease in humans and in mouse models. The severe neurodegeneration and neuroinflammation that occur in mnd2 (motor neuron degeneration 2) mice result from loss of the protease activity of Omi by the point mutation S276C; however, the substrates of Omi that induce neurodegeneration are unknown. We showed that Omi was required for the production of inflammatory molecules by microglia, which are the resident macrophages in the central nervous system. Omi suppressed the activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 and 2 (ERK1/2) by cleaving the upstream kinase MEK1 (mitogen-activated or extracellular signal-regulated protein kinase kinase 1). Knockdown of Omi in microglial cell lines led to activation of ERK1/2 and resulted in degradation of IκBα [α inhibitor of nuclear factor κB (NF-κB)], resulting in NF-κB activation and the expression of genes encoding inflammatory molecules, such as tumor necrosis factor-α and inducible nitric oxide synthase. The production of inflammatory molecules induced by the knockdown of Omi was blocked by the MEK1-specific inhibitor U0126. Furthermore, expression of the protease-deficient S276C Omi mutant in a microglial cell line had no effect on MEK1 cleavage or ERK1/2 activation. In the brains of mnd2 mice, we observed increased transcription of several genes encoding inflammatory molecules, as well as activation of astrocytes and microglia. Therefore, our study demonstrates that Omi is an intrinsic cellular factor that inhibits neuroinflammation.

Topics: Animals; Blotting, Western; Butadienes; Cell Line; Cell Line, Tumor; Enzyme Inhibitors; High-Temperature Requirement A Serine Peptidase 2; Humans; I-kappa B Proteins; Inflammation; MAP Kinase Kinase 1; Mice; Mice, Inbred C57BL; Microglia; Mitochondrial Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurodegenerative Diseases; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase Type II; Nitriles; Point Mutation; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Serine Endopeptidases; Signal Transduction; Tumor Necrosis Factor-alpha

Most therapeutic treatments for autoimmune arthritis rely on immunosuppressive drugs, which have side effects. Although a previous study by our group showed that specific ERK activation suppressed immune responses, its application in a therapeutic setting has never been tested. The aim of the present study was to define the ERK-dependent immunosuppressive mechanisms and to apply selective ERK activation for the treatment of experimental inflammatory arthritis.. A constitutively active ERK activator was coexpressed with a model antigen using lentivectors. Immunosuppressive mechanisms were characterized at the level of dendritic cell (DC) function, differentiation of antigen-specific Treg cells, and inhibition of inflammatory T cells. Administration of the ERK activator with antigen as a strategy to suppress inflammatory arthritis was tested in an experimental mouse model.. Selective ERK activation induced mouse and human DCs to secrete bioactive transforming growth factor β, a process required for suppression of T cell responses and differentiation of antigen-specific Treg cells. Treg cells strongly proliferated after antigen reencounter in inflammatory conditions, and these cells exhibited antigen-dependent suppressive activities. Inflammatory arthritis was effectively inhibited through antigen-specific mechanisms. Importantly, this strategy did not rely on identification of the initiating arthritogenic antigen. Equivalent mechanisms were demonstrated in human monocyte-derived DCs, setting the scene for a possible rapid translation of this approach to patients with rheumatoid arthritis.. This strategy of selective ERK activation resulted in an effective therapeutic protocol, with substantial advantages over DC or T cell vaccination.

Topics: Analysis of Variance; Animals; Arthritis, Experimental; Butadienes; Cell Differentiation; Cell Line; Cells, Cultured; Dendritic Cells; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Flow Cytometry; Humans; Immune Tolerance; Inflammation; Mice; Nitriles; T-Lymphocytes, Regulatory

Heat shock protein 47 (Hsp47), a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. Heat shock protein 47 is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare Hsp47 expression in normal gingival tissues and cyclosporine A-induced gingival overgrowth specimens and further explore the potential mechanisms that may lead to induction of Hsp47 expression.. Fifteen cyclosporine A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Western blot was used to investigate the effects of cyclosporine A on the expression of Hsp47 in human gingival fibroblasts. In addition, Aggregatibacter actinomycetemcomitans, interleukin-1 alpha (IL-1 alpha) and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 were added to seek the possible regulatory mechanisms of Hsp47 expression.. A significantly higher percentage of cells positively stained for Hsp47 was noted in the cyclosporine A-induced gingival overgrowth group than in the normal gingival group (p < 0.05). Expression of Hsp47 was observed mainly in the cytoplasm of fibroblasts, endothelial cells, epithelial cells and inflammatory cells. Expression of Hsp47 was significantly higher in cyclosporine A-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). Cyclosporine A upregulated Hsp47 expression in human gingival fibroblasts in a dose-dependent manner (p < 0.05). The addition of A. actinomycetemcomitans or interleukin-1 alpha significantly increased Hsp47 expression compared with cyclosporine A alone (p < 0.05). The MEK inhibitor U0126 was found to inhibit cyclosporine A-induced Hsp47 expression (p < 0.05).. Expression of Hsp47 is significantly upregulated in cyclosporine A-induced gingival overgrowth specimens, and Hsp47 expression induced by cyclosporine A in fibroblasts may be mediated by the MEK signal transduction pathway. The expression of Hsp47 could be significantly enhanced by A. actinomycetemcomitans and interleukin-1 alpha.

Topics: Aggregatibacter actinomycetemcomitans; Blotting, Western; Butadienes; Cells, Cultured; Cyclosporine; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Inhibitors; Epithelial Cells; Fibroblasts; Gingiva; Gingival Overgrowth; HSP47 Heat-Shock Proteins; Humans; Immunohistochemistry; Immunosuppressive Agents; Inflammation; Interleukin-1alpha; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Nitriles; Up-Regulation

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

We have identified a novel anti-inflammatory signaling pathway that leads to the expression of heme oxygenase-1 (HO-1) in response to bisdemethoxycurcumin (BDMC), an analog of curcumin. Treatment with BDMC suppressed inducible nitric oxide synthase expression and nitric oxide (NO) production by down-regulating NF-kappaB activity in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. These effects were reversed by blocking HO-1 activity or expression. The signaling pathway involved in BDMC-mediated HO-1 induction included Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase 1/2 (ERK1/2). BDMC induced phosphorylation of ERK1/2 in a CaMKII-dependent manner. Pretreatment with the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor, U0126, inhibited CaMKII-induced stimulation of HO-1 promoter activity, suggesting that ERK1/2 is a downstream mediator of CaMKII in BDMC signaling to HO-1 expression. Furthermore, the CaMKII-ERK1/2 cascade targets the transcription factor, NF-E2-related factor-2 (Nrf2). Finally, inhibition of the Ca(2+)-CaMKII-ERK1/2-linked cascade attenuated significantly suppression by BDMC of LPS-induced iNOS expression and subsequent NO production. Collectively, our findings identify a Ca(2+)/calmodulin-CaMKII-ERK1/2-Nrf2 cascade as a novel anti-inflammatory pathway mediating BDMC signaling to HO-1 expression in macrophages.

Topics: Animals; Butadienes; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line; Curcumin; Diarylheptanoids; Gene Expression Regulation, Enzymologic; Heme Oxygenase-1; Inflammation; Lipopolysaccharides; Macrophages; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-E2-Related Factor 2; NF-kappa B; Nitriles; Signal Transduction; Up-Regulation

Ligation of the leukotriene B(4) (LTB(4)) receptor 1 on effector memory CD8(+) T cells by LTB(4) is important for the recruitment of CD8(+) T cells into the airways, which appears central to the induction of airway hyperresponsiveness (AHR) and allergic inflammation. Phosphorylation of extracellular signal-regulated kinase (ERK) is important in activation and cytokine production from many cell types.. The roles of ERKs in effector CD8(+) T-cell function and on CD8(+) T cell-mediated AHR were determined.. Effector CD8(+) T cells were generated from OVA(257-264) (SIINFEKL) peptide-primed mononuclear cells from OT-1 mice. The effects of U0126, an ERK inhibitor, on effector CD8(+) T-cell function and on CD8(+) T cell-mediated AHR and allergic inflammation were examined.. Pretreatment of effector CD8(+) T cells with U0126 suppressed anti-CD3/anti-CD28-induced ERK1/2 phosphorylation and cytokine production, but did not affect LTB(4)-induced Ca(2+) mobilization or chemotaxis. Adoptive transfer of U0126-treated CD8(+) T cells into sensitized mice before secondary allergen challenge resulted in significant decreases in AHR, eosinophilic inflammation, goblet cell metaplasia, and IL-5 and IL-13 levels in bronchoalveolar lavage fluid of recipient mice. The number of transferred CD8(+) T cells accumulating in bronchoalveolar lavage fluid or lungs was unaffected by treatment.. ERK1/2-dependent pathways are essential for the effector functions of CD8(+) T cells, including T(H)2 cytokine production, allergic inflammation, and development of AHR. Inhibition of ERK1/2 signaling has potential therapeutic benefit in preventing CD8(+) T cell-mediated AHR.

Topics: Adoptive Transfer; Allergens; Animals; Bronchoalveolar Lavage Fluid; Butadienes; Calcium; CD8-Positive T-Lymphocytes; Chemotaxis; Enzyme Inhibitors; Eosinophils; Goblet Cells; Immunologic Memory; Inflammation; Leukotriene B4; Lung; MAP Kinase Signaling System; Metaplasia; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Receptors, Leukotriene B4; Respiratory Hypersensitivity; Th2 Cells

The pathogenesis of chronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation associated with lung neutrophilia and elevated levels of pro-inflammatory mediators in the bronchoalveolar lavage fluid or sputum of patients. Recent findings revealed that mitogen-activated protein kinase (MAPK) signaling cascade is involved in the inflammatory response of lung injury. In the present study we could elucidate the role of extracellular signal-related MAPK in the murine model of LPS-induced acute lung injury by using U0126, a specific inhibitor of MEK1/2, upstream kinases of ERK. Phosphorylation of ERK was inhibited by U0126 in vivo as well as in vitro. In freshly isolated human peripheral blood mononuclear cells U0126 dose-dependently blocked the release of IL-2 and TNF-alpha. For in vivo studies mice were exposed to aerosolized LPS to induce an acute lung injury mimicking some aspects of COPD. This led to a recruitment of neutrophils to the lung and to the release of pro-inflammatory cytokines into bronchoalveolar lavage. Pretreatment of mice with U0126 significantly reduced lung neutrophilia and diminished levels of TNF-alpha and chemotactic MIP-2 and KC in bronchoalveolar fluid. U0126 also decreased albumin levels in BAL fluid, a marker of vascular leakage. Histological examination of lung tissues revealed that ERK MAPK inhibition using U0126 efficiently attenuated LPS-induced pulmonary inflammatory responses. These data suggest that ERK signaling plays an important role in acute lung injury and pharmacologic inhibition of ERK provides a promising new therapeutic strategy for lung inflammatory diseases and in particular COPD.

Topics: Animals; Butadienes; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Humans; Inflammation; Leukocytes, Mononuclear; Lipopolysaccharides; Lung Injury; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Nitriles

The purpose of the present study was to investigate the roles of caspase 1 and extracellular signal-regulated kinase (ERK) in inflammation-induced inhibition of lacrimal gland secretion.. Lacrimal gland inflammation was induced by injection of lipopolysaccharide (LPS; to study the role of caspase 1) or IL-1beta (to study the role of ERK). Lacrimal gland protein secretion was measured using a spectrofluorometric assay. Caspase 1 and ERK activities in the lacrimal gland were measured by immunohistochemistry, Western blotting, or both. Aqueous tear production was measured using phenol red-impregnated cotton threads.. Injection of LPS into the lacrimal gland inhibited neurally and adrenergic agonist-induced protein secretion by 77% and 54%, respectively, and activated caspase 1. The degree of inhibition achieved by LPS was similar to that obtained with injection of IL-1beta. Inhibition of caspase 1 alleviated the inhibitory effect of LPS on lacrimal gland secretion. IL-1beta activated ERK in the lacrimal gland in vitro and in vivo, and this effect was blocked by UO126, an inhibitor of MEK, the ERK-activating enzyme. IL-1beta injection into the lacrimal gland inhibited aqueous tear production by 52% and inhibited neurally and adrenergic agonist-induced protein secretion by 80% and 55%, respectively. UO126 alleviated the inhibitory effect of IL-1beta on aqueous tear production and lacrimal gland protein secretion.. LPS inhibits lacrimal gland secretion by activating caspase 1, and IL-1beta activates the ERK pathway to inhibit lacrimal gland protein secretion and aqueous tear production.

Topics: Animals; Blotting, Western; Butadienes; Caspase 1; Dacryocystitis; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Eye Proteins; Female; Fluorescent Antibody Technique, Indirect; Inflammation; Interleukin-1beta; Lacrimal Apparatus; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Nitriles; Recombinant Proteins; Spectrometry, Fluorescence; Tears

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

Topics: Animals; Bee Venoms; Butadienes; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Hyperalgesia; Hypersensitivity; Imidazoles; Immunohistochemistry; Inflammation; Male; Mitogen-Activated Protein Kinase 3; Nitriles; p38 Mitogen-Activated Protein Kinases; Pain Threshold; Pyridines; Rats; Rats, Sprague-Dawley; Spinal Cord

Since serum IGF-I levels are related to risks of prostate cancer and cytokines like interleukin (IL)-6 and IL-8 have been implicated in prostate cancer progression, we investigated the effects of IGF-I on IL-6 and IL-8 expression in the prostate cancer cell.. In order to address the regulation by IGF-I of cytokine expression in prostate cancer cells we used cell cultures of established androgen dependent (LNCaP) and androgen-independent cell lines (DU-145 and PC-3).. We found that IGF-I stimulates IL-8 mRNA expression and secretion in DU-145 cells, whereas the secretion of IL-6 was hardly affected. IGF-I enhances IL-8 expression in synergy with IL-1beta, but not with tumour necrosis factor (TNF)alpha. Similarly, on IL-8 promoter activity, IGF-I exerted synergistic effects with IL-1beta, but not with TNFalpha. Although IGF-I stimulated the phosphorylation of both Akt (protein kinase B) and extracellular-regulated kinase (ERK), the effect of IGF-I at IL-8 expression was inhibited only by U0126, a pharmacological inhibitor of MAPK/ERK kinase (MEK) and not by inhibition of the upstream activator of Akt, phosphatidylinositol-3 kinase (PI3K).. Our results indicate that IGF-I stimulates IL-8 expression through the MEK-ERK pathway in DU-145 cells, at least in part, by augmentation of transcriptional activity. This finding is in accordance with our observations that IGF-I did not influence cytokine secretion and phosphorylation of ERK in LNCaP or PC-3 cells. It remains to be established whether IL-8 mediates certain effects of IGF-I on prostate cancer cells and whether differential responsiveness of prostate cancer cells to IGF-I relates to certain stages of prostate cancer.

Topics: Androstadienes; Butadienes; Cell Line, Tumor; Cytokines; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Insulin-Like Growth Factor I; Interleukin-6; Interleukin-8; Male; Nitriles; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Neoplasms; Recombinant Proteins; Transfection; Wortmannin

Vein graft failure following bypass surgery is a frequent and important clinical problem. The vascular injury caused by arterialization is responsible for vein graft intimal hyperplasia, a lesion generated by medial smooth muscle cell proliferation and migration into the intima, increased extracellular matrix deposition, and formation of a thick neointima. Development of the neointima into a typical atherosclerotic lesion and consequent stenosis ultimately result in vein graft failure. Endothelial damage, inflammation, and intracellular signaling through mitogen-activated protein kinases (MAPKs) have been implicated in the early stages of this process. We therefore investigated the effects of topical inhibition of ERK-1/2 MAPK activation on vascular cell proliferation and apoptosis, and on the inflammatory response in a canine model of vein graft arterialization. For this purpose, vein grafts were incubated with the MEK-1/2 inhibitor, UO126, ex vivo for 30 min before grafting. This treatment effectively abolished arterialization-induced ERK-1/2 activation, decreased medial cell proliferation, and increased apoptosis. UO126 treatment also inhibited the vein graft infiltration by myeloperoxidase-positive inflammatory cells that follows vein graft arterialization. Thus, topical ex vivo administration of MAPK inhibitors can provide a pharmacological tool to prevent or reduce the vascular cell responses that lead to vein graft intimal hyperplasia and graft failure.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents; Apoptosis; Butadienes; Carotid Arteries; Cell Proliferation; Dogs; Inflammation; Jugular Veins; Mitogen-Activated Protein Kinases; Models, Animal; Nitriles

Numerous studies have implicated spinal extracellular signal-regulated kinases (ERKs) as mediators of nociceptive plasticity. These studies have utilized pharmacological inhibition of MEK to demonstrate a role for ERK signaling in pain, but this approach cannot distinguish between effects of ERK in neuronal and non-neuronal cells. The present studies were undertaken to test the specific role of neuronal ERK in formalin-induced inflammatory pain. Dominant negative MEK (DN MEK) mutant mice in which MEK function is suppressed exclusively in neurons were tested in the formalin model of inflammatory pain.. Formalin-induced second phase spontaneous pain behaviors as well as thermal hyperalgesia measured 1 - 3 hours post-formalin were significantly reduced in the DN MEK mice when compared to their wild type littermate controls. In addition, spinal ERK phosphorylation following formalin injection was significantly reduced in the DN MEK mice. This was not due to a reduction of the number of unmyelinated fibers in the periphery, since these were almost double the number observed in wild type controls. Further examination of the effects of suppression of MEK function on a downstream target of ERK phosphorylation, the A-type potassium channel, showed that the ERK-dependent modulation of the A-type currents is significantly reduced in neurons from DN MEK mice compared to littermate wild type controls.. Our results demonstrate that the neuronal MEK-ERK pathway is indeed an important intracellular cascade that is associated with formalin-induced inflammatory pain and thermal hyperalgesia.

Topics: Animals; Behavior, Animal; Butadienes; Enzyme Activation; Formaldehyde; Genes, Dominant; Hot Temperature; Hyperalgesia; Inflammation; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase Kinases; Neurons; Nitriles; Pain; Potassium Channels

Nitric oxide (NO) from astrocytes is one of the signalers used by the brain's extensive glial-neuronal-vascular network, but its excessive production by pro-inflammatory cytokine-stimulated glial cells can be cytodestructive. Here, we show how three pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) together stimulated the activation, but not the prior expression, of NOS-2 protein via a mechanism involving MEK-ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS-2 mRNA which were followed by bursts of MEK-ERK activities, synthesis of the NOS-2 co-factor tetrahydrobiopterin (BH(4)), a build-up of NOS-2 protein and from it active NOS-2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS-2 expression nor NOS-2 protein accumulation but stopped BH(4) synthesis and the assembly of the NOS-2 protein into active NOS-2 enzyme. The complete blockage of active NOS-2 production by the brief exposure to U0126 was bypassed by simply adding BH(4) to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS-2 gene expression and accumulation of NOS-2 protein and the second being the synthesis of the BH(4) factor needed to dimerize the NOS-2 protein into active, NO-making NOS-2 enzyme.

Topics: Astrocytes; Biopterins; Butadienes; Cells, Cultured; Cytokines; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Gene Expression Regulation, Enzymologic; Humans; Inflammation; Inflammation Mediators; Interferon-gamma; Interleukin-1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitriles; Proto-Oncogene Proteins c-raf; Tumor Necrosis Factor-alpha

Although inflammation and apoptosis are known to play important roles in cisplatin nephrotoxicity, the exact intracellular signaling mechanisms are not well understood. Recent reports that extracellular signal-regulated kinase (ERK1/2) pathway mediates cisplatin-induced caspase activation and apoptosis in cultured renal tubular cells led us to investigate the effect of MAPK/ERK kinase (MEK) inhibitor, an immediate upstream of ERK1/2 in cisplatin-induced acute renal failure (ARF) in mice.. The effect of MEK/ERK1/2 inhibition on kidney tumor necrosis factor-alpha (TNF-alpha (gene expression, inflammation, the activation of tissue caspases, and apoptosis were examined in addition to its effects on renal function and histology in cisplatin-induced ARF in mice.. Pretreatment of MEK inhibitor, U0126, decreased ERK1/2 phosphorylation following cisplatin administration with significant functional and histologic protection. This beneficial effect was accompanied by decrease in TNF-alpha gene expression level and inflammation, as well as in caspase 3 activity and apoptosis.. These data provide evidence that ERK1/2 pathway functions as an upstream signal for TNF-alpha-mediated inflammation and caspase 3-mediated apoptosis in cisplatin-induced ARF in mice and suggest that ERK1/2 can be a novel therapeutic target in cisplatin nephrotoxicity.

Topics: Animals; Antineoplastic Agents; Apoptosis; Butadienes; Caspases; Cisplatin; Enzyme Inhibitors; Inflammation; Kidney; Male; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; RNA, Messenger; Tumor Necrosis Factor-alpha

Interleukin (IL)-13 receptor activation on airway smooth muscle cells induces eotaxin release and activates multiple signaling pathways including mitogen-activated protein kinases, and signal transducer and activator of transcription 6 (STAT6). To examine a requirement for STAT6 in mediating IL-13-stimulated eotaxin release we used antisense oligodeoxynucleotides (ODNs) to downregulate endogenous STAT6 protein. STAT6 antisense ODNs were taken up by about 85% of cells. Selective downregulation of STAT6 protein occurred with antisense ODNs, but not with sense or scrambled ODNs. Eotaxin release induced by IL-13 or IL-4 (10 ng/ml) was reduced by 81 +/- 4 and 75 +/- 7%, respectively, in cells transfected with antisense ODNs (p < 0.001), but not with a sense ODN or a scrambled ODN. Eotaxin release induced by IL-1beta was unaffected by STAT6 antisense ODN (p > 0.05). Finally, IL-13- or IL-4-dependent eotaxin release was abolished when inhibitors of both p42/p44 ERK (U0126, 10 microM) and p38 (SB202190, 10 microM) mitogen-activated protein kinase pathways were combined in STAT6 antisense ODN-transfected cells. In contrast, about 25% of the response remained when each inhibitor was examined alone in STAT6 antisense ODN-treated cells. These data support roles for both STAT6- and mitogen-activated protein kinase-dependent pathways in mediating eotaxin release from airway smooth muscle by IL-13 or IL-4.

Topics: Adult; Aged; Asthma; Bronchial Hyperreactivity; Butadienes; Cells, Cultured; Chemokine CCL11; Chemokines; Chemokines, CC; Down-Regulation; Female; Humans; Imidazoles; Inflammation; Interleukin-13; Interleukin-4; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Muscle, Smooth; Nitriles; Oligodeoxyribonucleotides, Antisense; p38 Mitogen-Activated Protein Kinases; Pyridines; Respiratory Mucosa; Signal Transduction; STAT6 Transcription Factor; Trans-Activators

The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-beta (TGF-beta) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-beta-induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-beta-induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-beta and BNP for 24 and 48 hours. TGF-beta, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-beta effects; 88% and 85% of all TGF-beta-regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-beta-regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (alpha-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNFalpha-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate-dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-beta-induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.

Topics: Adolescent; Blotting, Western; Butadienes; Cell Division; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Flavonoids; Gene Expression Profiling; Gene Expression Regulation; Humans; Inflammation; Male; MAP Kinase Signaling System; Middle Aged; Muscle Proteins; Natriuretic Peptide, Brain; Nitriles; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Ventricular Remodeling

Tissue factor (TF) is the main initiator of coagulation in vivo. Recently, however, a role for TF as a cell receptor involved in signal transduction has been suggested. The aim of the present study was to assess whether activated factor VII (FVIIa) binding to TF could induce smooth muscle cell (SMC) proliferation and to clarify the possible intracellular mechanism(s) responsible for this proliferation.. Cell proliferation was induced by FVIIa in a dose-dependent manner, as assessed by [3H]thymidine incorporation and direct cell counting, whereas no response was observed with active site-inhibited FVIIa (FVIIai), which is identical to FVIIa but is devoid of enzymatic activity. Similarly, no proliferation was observed when binding of FVIIa to TF was prevented by the monoclonal anti-TF antibody AP-1. Activation of the p44/42 mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinases 1 and 2 [ERK 1/2]) pathway on binding of FVIIa to TF was demonstrated by transient ERK phosphorylation in Western blots and by suppression of proliferation with the specific MEK (MAP kinase/ERK kinase) inhibitor UO126. ERK phosphorylation was not observed with FVIIai or when cells were pretreated with AP-1.. These data indicate a specific effect by which binding of FVIIa to TF on the surface of SMCs induces proliferation via a coagulation-independent mechanism and possibly indicate a new link between coagulation, inflammation, and atherosclerosis.

Topics: Animals; Antibodies, Monoclonal; Aorta, Thoracic; Arteriosclerosis; Binding Sites; Blood Coagulation; Butadienes; Cell Division; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Factor VIIa; Humans; Inflammation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitriles; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Rabbits; Recombinant Fusion Proteins; Signal Transduction; Thromboplastin

Group I metabotropic glutamate receptors (mGluRs) and their downstream signaling pathways, which involve the extracellular signal-regulated kinases (ERKs), have been implicated as mediators of plasticity in several pain models. In this study, we report that inflammation leads to a long-lasting enhancement of behavioral responses induced by activation of spinal group I mGluRs. Thus, the nocifensive response to intrathecal injection of the group I mGluR agonist (RS)-3,5-Dihydroxyphenylglycine (DHPG) is significantly potentiated seven days following Complete Freund's Adjuvant (CFA)-induced inflammation of the hind paw. This potentiation is not associated with increased mGlu1 or mGlu5 receptor expression but is associated with increased levels of phosphorylated ERK in dorsal horn neurons. We also tested whether the increased behavioral response to DHPG following inflammation may be explained by enhanced coupling of the group I mGluRs to ERK activation. DHPG-induced ERK phosphorylation in the dorsal horn is not potentiated following inflammation. However, inhibiting ERK activation using a MEK inhibitor, U0126, following inflammation attenuates the intrathecal DHPG-induced behavioral responses to a greater extent than in control animals. The results from this study indicate that persistent ERK activation is required for the enhanced behavioral responses to spinal group I mGluR activation following inflammation and suggest that tonic modulation of ERK activity may underlie a component of central sensitization in dorsal horn neurons.

Topics: Animals; Animals, Outbred Strains; Behavior, Animal; Butadienes; Enzyme Inhibitors; Freund's Adjuvant; Inflammation; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; Nitriles; Nociceptors; Pain; Posterior Horn Cells; Receptors, Metabotropic Glutamate

The effects of OSM on proliferation and differentiation of osteosarcoma and nontransformed osteoblasts were analyzed. OSM downregulates osteoblast markers but induces the glial fibrillary acidic protein by the combined activation of PKCdelta and STAT3, offering new lines of therapeutic investigations.. Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family implicated in embryonic development, differentiation, inflammation, and regeneration of various tissues, mainly the liver, bone, and the central nervous and hematopoietic systems. One particularity of OSM relies on its growth inhibitory and pro-differentiating effects on a variety of tumor cell lines such as melanoma, providing arguments for a therapeutic application of OSM. The objective of this study was to analyze the effects of OSM on osteosarcoma cell lines proliferation and differentiation.. Proliferation was analyzed by 3H thymidine incorporation. Differentiation was analyzed by semiquantitative RT-PCR and immunocytochemistry for various markers. Alizarin red S staining was used to evaluate bone nodule formation. Morphological changes were studied by confocal and electron microscopy. Western blotting, kinases inhibitors, and dominant negative STAT3 were used to identified the signaling pathways implicated.. OSM inhibits the growth of rat osteosarcoma cell lines as well as normal osteoblasts, in correlation with induction of the cyclin-dependent kinases inhibitor p21WAF1. However, OSM reduces osteoblast markers such as alkaline phosphatase, osteocalcin, and bone sialoprotein, leading to strong inhibition of mineralized nodule formation. This inhibitory effect is restricted to mature osteoblasts and differentiated osteosarcoma because OSM effectively stimulates osteoblast markers and bone nodule formation in early, but not late, bone marrow mesenchymal stem cell (BMSC) cultures. In osteosarcoma cells or BMSC, OSM induces expression of the glial fibrillary acidic protein (GFAP) as well as morphological and ultrastructural changes, for example, elongated shape and bundles of microfilaments in cell processes. Rottlerin (PKCdelta inhibitor), and to a lesser degree UO126 (MEK/ERK inhibitor), prevents the loss of osteoblastic markers by OSM, whereas dominant negative STAT3 prevents GFAP induction.. These results highlight the particular gene expression profile of OSM-treated osteosarcoma cells and BMSCs, suggesting either a osteocytic or a glial-like phenotype. Together with the implication of PKCdelta, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.

Topics: Alkaline Phosphatase; Animals; Anthraquinones; Blotting, Western; Bone and Bones; Bone Marrow Cells; Butadienes; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; DNA; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Glial Fibrillary Acidic Protein; Immunohistochemistry; Inflammation; Integrin-Binding Sialoprotein; Interleukin-6; Mesoderm; Microscopy, Confocal; Microscopy, Electron; Models, Biological; Nitriles; Osteoblasts; Osteocalcin; Osteosarcoma; Protein Kinase C; Protein Kinase C-delta; Rats; Regeneration; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Signal Transduction; STAT3 Transcription Factor; Stem Cells; Thymidine; Time Factors; Trans-Activators; Transfection

To investigate the intracellular signal transduction pathways involved in regulating the gene expression of brain-derived neurotrophic factor (BDNF) in primary afferent neurons, we examined the activation of extracellular signal-regulated protein kinase (ERK) in dorsal root ganglion (DRG) neurons after peripheral inflammation and sciatic nerve transection. Peripheral inflammation induced an increase in the phosphorylation of ERK, mainly in tyrosine kinase A-containing small-to-medium-diameter DRG neurons. The treatment of the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126 reversed the pain hypersensitivity and the increase in phosphorylated-ERK (p-ERK) and BDNF in DRG neurons induced by complete Freund's adjuvant. On the other hand, axotomy induced the activation of ERK mainly in medium-and large-sized DRG neurons and in satellite glial cells. U0126 suppressed the axotomy-induced autotomy behavior and reversed the increase in p-ERK and BDNF. The intrathecal application of nerve growth factor (NGF) induced an increase in the number of p-ERK-and BDNF-labeled cells, mainly small neurons, and the application of anti-NGF induced an increase in p-ERK and BDNF in some medium-to-large-diameter DRG neurons. The activation of MAPK in the primary afferents may occur in different populations of DRG neurons after peripheral inflammation and axotomy, respectively, through alterations in the target-derived NGF. These changes, including the changes in BDNF expression, might be involved in the pathophysiological changes in primary afferent neurons.

Topics: Activating Transcription Factor 3; Animals; Axotomy; Behavior, Animal; Brain-Derived Neurotrophic Factor; Butadienes; Enzyme Activation; Enzyme Inhibitors; Ganglia, Spinal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Immunohistochemistry; Inflammation; Injections, Spinal; Male; Mitogen-Activated Protein Kinases; Neurofilament Proteins; Neuroglia; Neurons, Afferent; Nitriles; Pain Threshold; Phosphorylation; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Transcription Factors

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.

Topics: Amino Acid Chloromethyl Ketones; Antigens, CD; Apoptosis; Butadienes; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; fas Receptor; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrogen-Ion Concentration; Imidazoles; Inflammation; Interferon-gamma; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Necrosis; NF-kappa B; Nitriles; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Pyridines; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; T-Lymphocytes; Tumor Necrosis Factor-alpha

Activation of ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinase in dorsal horn neurons of the spinal cord by peripheral noxious stimulation contributes to short-term pain hypersensitivity. We investigated ERK activation by peripheral inflammation and its involvement in regulating gene expression in the spinal cord and in contributing to inflammatory pain hypersensitivity. Injection of complete Freund's adjuvant (CFA) into a hindpaw produced a persistent inflammation and a sustained ERK activation in neurons in the superficial layers (laminae I-IIo) of the dorsal horn. CFA also induced an upregulation of prodynorphin and neurokinin-1 (NK-1) in dorsal horn neurons, which was suppressed by intrathecal delivery of the MEK (MAP kinase kinase) inhibitor U0126. CFA-induced phospho-ERK primarily colocalized with prodynorphin and NK-1 in superficial dorsal horn neurons. Although intrathecal injection of U0126 did not affect basal pain sensitivity, it did attenuate both the establishment and maintenance of persistent inflammatory heat and mechanical hypersensitivity. Activation of the ERK pathway in a subset of nociceptive spinal neurons contributes, therefore, to persistent pain hypersensitivity, possibly via transcriptional regulation of genes, such as prodynorphin and NK-1.

Topics: Animals; Butadienes; Disease Models, Animal; Enkephalins; Enzyme Activation; Enzyme Inhibitors; Freund's Adjuvant; Hindlimb; Hyperalgesia; Inflammation; Injections, Spinal; Male; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Pain; Posterior Horn Cells; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Substance P; Up-Regulation