calcimycin and phorbolol-myristate-acetate

Inflammation‑associated damage may occur in any tissue following infection, exposure to toxins, following ischemia, and in allergic and auto‑immune reactions. Inflammation may also result from mast cell degranulation induced by the intracellular calcium concentration. The inflammatory process may be inhibited by compounds that affect mast cells. Bisdemethoxycurcumin [1,7‑bis(4‑hydroxyphenyl) hepta‑1,6‑diene‑3,5‑dione, BDCM] is the active component of turmeric. It has anticancer, antioxidant and antibacterial properties. To investigate the molecular mechanism associated with the anti‑inflammatory activity of BDCM, human mast cell line 1 (HMC‑1) cells were treated with phorbol‑12‑myristate‑13‑acetate (PMA) and calcium ionophore A23187 (A23187) to induce the inflammatory process. Various HMC‑1 cells were pretreated with BDCM prior to stimulation of inflammation. BDCM inhibited the inflammation‑triggered production of cytokines including interleukin (IL)‑6, IL‑8, and tumor necrosis factor (TNF)‑α. BDCM inhibition extended to the gene level. In activated HMC‑1 cells, phosphorylation levels of extracellular signal‑regulated kinase, c‑jun N‑terminal kinase and p38 mitogen‑activated protein kinase were decreased by treatment with BDCM. BDCM also inhibited nuclear factor‑(NF)‑κB activation and IκB degradation. In conclusion, BDCM suppresses the expression of TNF‑α, IL‑8, and IL‑6 by inhibiting the NF‑κB and mitogen activated protein kinase signaling pathways.

Topics: Calcimycin; Cell Line; Curcumin; Diarylheptanoids; Gene Expression Regulation; Humans; Inflammation; Mast Cells; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor RelA

Geraniol is a monoterpene alcohol that has anti-fungal, anti-cancer and anti-nociceptive properties, but its anti-allergic rhinitis (AR) property is unclear.. In this study, the anti-inflammatory role and its possible mechanisms of geraniol in human mast cell line (HMC-1) cells stimulated by inflammatory trigger phorbol 12-myristate 13-acetate plus A23187 (PMACI), as well as in ovalbumin (OVA)-induced AR mice models were investigated.. PMACI results in a significant increase in the production of proinflammatory cytokines, such as TNF-α, IL-1β, MCP-1, IL-6 and as well as histamine. Geraniol was found to inhibit both TNF-α, IL-1β and IL-6 protein and mRNA expressions at concentrations of 40, 80, 160 μM. In OVA-induced AR models, geraniol treatment was able to suppress AR biomarkers (OVA-specific IgE and IL-1β as well as histamine) and nasal rub scores. Interestingly, p38, a member of the mitogen-activated protein kinase (MAPK) signaling family, was found to be increasingly hypophosphorylated as geraniol dose was increased. Similar decreases in the nuclear level of p65, a member of the nuclear factor kappa B (NF-κB) signaling pathway, were also observed.. Our data highlights that the anti-inflammatory properties of geraniol on AR-related markers in activated HCM-1 cells and OVA-induced AR models may be mediated through the regulation of the MAPK/NF-κB signaling pathway.

Topics: Acyclic Monoterpenes; Animals; Anti-Allergic Agents; Anti-Inflammatory Agents, Non-Steroidal; Calcimycin; Cell Line; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Histamine; Humans; Inflammation Mediators; Mice; Mice, Inbred BALB C; Molecular Structure; Ovalbumin; Rhinitis, Allergic; Structure-Activity Relationship; Terpenes; Tetradecanoylphorbol Acetate

Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin,

Topics: Calcimycin; Candida albicans; Extracellular Traps; Granulomatous Disease, Chronic; Healthy Volunteers; Humans; Metabolic Networks and Pathways; Neutrophils; Nigericin; Streptococcus; Tetradecanoylphorbol Acetate

Mast cells are central players in immediate-type hypersensitvity and inflammatory responses. In the present study, the effects of britanin on the passive cutaneous anaphylaxis (PCA) reaction in mice and on the phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-induced production of pro-inflammatory cytokines in human mast cell line (HMC-1) were evaluated. The oral administration of britanin (10-20 mg/kg) decreased the mast cell-mediated PCA reaction in IgE-sensitized mice. In the activity and mechanism of britanin in vitro assay, britanin suppressed the gene expression and secretion of pro-inflammatory cytokines in a dose-dependent manner in HMC-1. In addition, britanin attenuated PMACI-induced activation of NF-κB as indicated by the inhibition of the degradation of IκBα, nuclear translocation of NF-κB, NF-κB/DNA binding activity assay, and blocked the phosphorylation of p38 MAP kinase, in a dose-dependent manner. We conclude that britanin may have potential as a treatment for allergic-inflammatory diseases.

Topics: Administration, Ophthalmic; Animals; Calcimycin; Calcium Ionophores; Cells, Cultured; Cytokines; Dose-Response Relationship, Drug; Humans; Hypersensitivity, Immediate; Inflammation; Inflammation Mediators; Inula; Lactones; Male; Mast Cells; Mice, Inbred ICR; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Passive Cutaneous Anaphylaxis; Phosphorylation; Phytotherapy; Sesquiterpenes; Tetradecanoylphorbol Acetate

We have previously reported that acteoside inhibits the release of β-hexosaminidase from immunoglobulin E (IgE)-sensitized and bovine serum albumin-stimulated rat basophilic leukemia cells as well as the intracellular calcium level, release of histamine from, and production of tumor necrosis factor-alpha and interleukin-4 in human basophilic (KU812) cells. However, the molecular mechanism underlying the anti-allergic effects of acteoside has not yet been elucidated. Here, we used microarray analysis to determine the global gene expression profile of KU812 cells treated with acteoside and calcium ionophore A23187 plus phorbol-12-myristate 13-acetate (A23187+PMA), and the results were validated by real-time polymerase chain reaction (PCR) and Western blotting. Microarray analysis results showed that of the 201 genes in the microarray, 149 genes were up-regulated, while 52 genes were down-regulated. The significantly down-regulated genes have functions as chemokine and IgE receptors, as well as for immune response. Results of the validation of the microarray results using real-time PCR showed a significant decrease in the expressions of Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide (FCER1A) and nuclear factor of activated T cell, cytoplasmic, calcineurin-dependent 1 (NFATC1) genes. Furthermore, Western blotting showed a decrease in the phosphorylation of mitogen-activated protein kinase (MAPK) Jun N terminal kinase (JNK), revealing the role of JNK MAPK in acteoside-mediated allergy inhibition. We determined that the anti-allergy effects of acteoside were due to the down-regulation of the expressions of the chemokine ligand 1 (CCL1), CCL2, CCL3, CCL4, FCER1A and NFATC1 genes and the inhibition of the MAPK pathway through decreased JNK phosphorylation.

Topics: Animals; Anti-Allergic Agents; Basophils; Calcimycin; Calcium Ionophores; Cell Line; Cistanche; Down-Regulation; Glucosides; Humans; Hypersensitivity, Immediate; Interleukin-4; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; NFATC Transcription Factors; Phenols; Rats; Receptors, IgE; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Activation of mast cells in rheumatoid synovial tissue has often been associated with tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 production and disease pathogenesis by adjacent cell types. Butea monosperma (BM) is a well known medicinal plant in India and the tropics. The aim of this study was to examine whether a standardized extract of BM flower (BME) could inhibit inflammatory reactions in human mast cells (HMC) using activated HMC-1 cells as a model. Four previously characterized polyphenols--butrin, isobutrin, isocoreopsin, and butein--were isolated from BME by preparative thin layer chromatography, and their purity and molecular weights were determined by liquid chromatography/mass spectrometry analysis. Our results showed that butrin, isobutrin, and butein significantly reduced the phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced inflammatory gene expression and production of TNF-alpha, IL-6, and IL-8 in HMC-1 cells by inhibiting the activation of NF-kappaB. In addition, isobutrin was most potent in suppressing the NF-kappaB p65 activation by inhibiting IkappaBalpha degradation, whereas butrin and butein were relatively less effective. In vitro kinase activity assay revealed that isobutrin was a potent inhibitor of IkappaB kinase complex activity. This is the first report identifying the molecular basis of the reported anti-inflammatory effects of BME and its constituents butrin, isobutrin, and butein. The novel pharmacological actions of these polyphenolic compounds indicate potential therapeutic value for the treatment of inflammatory and other diseases in which activated mast cells play a role.

Topics: Butea; Calcimycin; Chalcones; Chromatography, Thin Layer; Enzyme-Linked Immunosorbent Assay; Flavonoids; Gas Chromatography-Mass Spectrometry; Gene Expression; Humans; I-kappa B Kinase; Inflammation; Interleukin-6; Interleukin-8; Mast Cells; NF-kappa B; Plant Extracts; Plants, Medicinal; Polymerase Chain Reaction; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system.. We show that VEGF is able to induce RCAN1.4 expression during cellular proliferation and differentiation, and that VEGF-mediated expression of RCAN1.4 was inhibited by the use of inhibitors to protein kinase C (PKC) and calcineurin. Further analysis revealed that siRNA silencing of PKC-delta expression partially inhibited VEGF-stimulated RCAN1.4 expression. Knockdown of RCAN1.4 with siRNA resulted in a decrease in cellular migration and disrupted tubular morphogenesis when HDMECs were either stimulated with VEGF in a collagen gel or in an endothelial/fibroblast co-culture model of angiogenesis. Analysis of intracellular signalling revealed that siRNA mediated silencing of RCAN1.4 resulted in increased expression of specific nuclear factor of activated T-cells (NFAT) regulated genes.. Our data suggests that RCAN1.4 expression is induced by VEGFR-2 activation in a Ca(2+) and PKC-delta dependent manner and that RCAN1.4 acts to regulate calcineurin activity and gene expression facilitating endothelial cell migration and tubular morphogenesis.

Topics: Anti-Bacterial Agents; Calcimycin; Calcineurin; Calcium; Cell Line; Cell Proliferation; Cells, Cultured; DNA-Binding Proteins; Endothelial Cells; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Muscle Proteins; Phosphorylation; Protein Kinase C-delta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Tetradecanoylphorbol Acetate; Vascular Endothelial Growth Factor A

Crosslinking of Fcvarepsilon receptor on mast cells induces IL-3 gene expression with the concentration dependent of intracellular calcium, but its regulatory mechanism remains unclear. Here, we found that phorbol 12-myristate 13-acetate (PMA) alone did not induce IL-3 gene expression, but potentiated A23187-induced IL-3 gene expression. Interestingly, the A23187-induced IL-3 promoter activity was suppressed by PMA, but it was enhanced when IL-3 promoter contained enhancer region, a DH site. While IL-3 mRNA expression was increased by A23187 and PMA in a dose-dependent manner, the promoter activity appeared all or none in all doses of A23187 and PMA. IL-3 promoter region between -293 and -150bp was responsible for A23187-induced gene expression and PMA- or cyclosporin A (CsA)-mediated suppression. Taken together, IL-3 gene expression was primarily regulated at the transcriptional level, which was differentially controlled by a restricted promoter and enhancer region.

Topics: Animals; Base Sequence; Calcimycin; Cyclosporine; Deoxyribonuclease I; Enhancer Elements, Genetic; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Interleukin-3; Mast Cells; Mice; Molecular Sequence Data; Promoter Regions, Genetic; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic

Artificial activation is required for successful intracytoplasmic sperm injection (ICSI) to induce haploidy pronuclear formation with extraction of second polar body. The present study showed that an additional treatment with Phorbol 12-myristate 13-acetate (PMA) followed by Ca(2+) ionophore treatment improved the rate of pronuclear formation, however, these oocytes had more than two pronuclei because of the suppression of polar body emission. The cultivation with MEK inhibitor U0126 followed by Ca(2+) ionophore also increased the rate of pronuclear formation but suppressed the emission of second polar body. These results suggested that the decrease of MAP kinase activity at early stage of artificial activation, concomitantly with decreasing p34(cdc2) kinase activity, prevented the second polar body extraction. We investigated that the timing of MAP kinase inactivation affected the extraction of the polar body and pronuclear formation rate. The addition of PMA 8 hr after Ca(2+) ionophore treatment induced the delay of MAP kinase inactivation, which resulted in haploidy pronuclear formation with emission of polar body. These results demonstrated for the first time that the delay of MAP kinase inactivation induced by PMA improved pronuclear formation with the extraction of second polar body in porcine oocytes activated by Ca(2+) ionophore. This method can be available for successfully ICSI in low response species of oocyte activation to Ca(2+) ionophore including pig.

Topics: Animals; Butadienes; Calcimycin; Calcium; CDC2 Protein Kinase; Female; Ionophores; Male; Mitogen-Activated Protein Kinases; Nitriles; Oocytes; Protein Kinase Inhibitors; Sperm Injections, Intracytoplasmic; Swine; Tetradecanoylphorbol Acetate; Time Factors

Gammi-danguieumja (GD) is clinically used in South Korea for treating atopic dermatitis. However, its effects in experimental models remain unknown. We investigated a possible effect of GD on cytokines production using human T cell line (MOLT-4) or human mast cell line. As a result, GD (0.01 mg/mL)-containing medium in stimulated culture supernatants increased IL-2 and IFN-gamma, and decreased IL-4 secretion in MOLT-4. GD (0.01-1 mg/mL)-containing medium in stimulated culture supernatants dose-dependently and significantly decreased IL-8, IL-13, and tumor necrosis factor-alpha secretion on the phorbol 12-myristate 13-acetate and A23187-stimulated HMC-1. In addition, GD inhibited histamine release from activated mast cells. These results suggest that GD contributes to the regulation of atopic allergic reactions.

Topics: Animals; Anti-Allergic Agents; Calcimycin; Cell Line; Cell Survival; Cytokines; Drugs, Chinese Herbal; Histamine Release; Humans; Interferon-gamma; Interleukin-8; Ionophores; Mast Cells; p-Methoxy-N-methylphenethylamine; Rats; Rats, Sprague-Dawley; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Osteoprotegerin (OPG), a secreted member of the tumor necrosis receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Parathyroid hormone (PTH), a potent inducer of osteoclast formation, suppresses OPG mRNA expression in vitro and in vivo. To determine the molecular basis of this inhibition, we analyzed the effects of PTH on the human OPG promoter (-5917 to +19) fused with beta-galactosidase reporter gene in stable and transient transfections into rat osteoblast-like UMR106 cells. The effect of PTH on OPG promoter expression was biphasic and concentration-dependent. PTH (1-100 nM) induced the transcriptional activity of the OPG promoter (1.7-fold) at 8 h followed by a gradual decrease with maximal inhibition (6.6-fold) at 24-48 h. To ascertain the signal transduction pathways mediating PTH (1-38) effects on OPG gene expression, we compared the effects of PTH with PTH analogs, parathyroid hormone-related protein 1-34 (PTHrP 1-34), forskolin, 3-isobutyl-1-methylxanthine (IBMX), dibutyryl cAMP, phorbol-12-myristate-13-acetate (PMA), thapsigargin and calcium ionophore A23187. PTH 1-31 and PTHrP 1-34, which stimulate the cAMP/PKA pathway, and other activators of cAMP/PKA, forskolin, IBMX, N(6), O(2')-dibityryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), all elicited a similar biphasic response on OPG promoter expression. PTH analogs PTH 3-34 and PTH 7-34, that do not stimulate cAMP production, had no effect on OPG expression. In contrast, phorbol-12-myristate-13-acetate (PMA), an activator of PKC, stimulated OPG promoter expression, while thapsigargin and calcium ionophore A23187, which increase intracellular Ca(2+), showed a dose-dependent inhibition of OPG promoter expression. To delineate the promoter sequences that mediate the inhibitory effects of PTH on OPG transcription, we analyzed systematic deletions of the OPG promoter for responsiveness in transient transfection assays. The major inhibitory effects of PTH were localized to 391 bp (-372 to +19) of the proximal promoter. Deletions of the promoter region led to a complete loss of responsiveness. Taken together, these results demonstrate that the inhibitory effects of PTH on OPG are mediated at the transcriptional level through cis elements in the proximal promoter. The similar biphasic response of OPG to PTH, PTH 1-31, PTHrP 1-34, forskolin, IBMX and dibutyryl cAMP suggests that PTH regulates OPG transcription via activation of the cAMP/PKA signal transduct

Topics: 1-Methyl-3-isobutylxanthine; Animals; Bucladesine; Calcimycin; Colforsin; Gene Expression; Glycoproteins; Osteoblasts; Osteoprotegerin; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Promoter Regions, Genetic; Proteins; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction; Tetradecanoylphorbol Acetate; Thapsigargin

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.

Topics: Adenosine Diphosphate; Calcimycin; Calcium; Cell Line; Enzyme Activation; Epinephrine; Glycerophospholipids; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Myristic Acid; Myristic Acids; Phosphatidic Acids; Phosphatidylinositols; Phospholipase D; Phospholipases; Platelet Activating Factor; Tetradecanoylphorbol Acetate; Thrombin; Tumor Cells, Cultured

Previous studies have shown that the glyceride derivative, sn-1,2-isopropylidene-3-decanoyl-glycerol (IpOCOC9), can trigger human leukocyte histamine release. Approximately 25% of the total cellular histamine content is extruded in the presence of 206 microM of IpOCOC9; at 69 microM, however, the secretagogue action of the compound is marginal. The characteristics of the release induced by IpOCOC9 are closely similar to those reportedly recorded at hyperosmolar triggering of basophils with mannitol, and in many respects they also mimic those observed at phorbol ester-induced histamine release. The compound decanoic acid cyclopentyl methylester (DACPME), a structural analogue of IpOCOC9, fails to induce histamine release. IpOCOC9, but not DACPME, stimulates human polymorphonuclear leukocyte cytosolic Ca2+- and phospholipid-dependent histone III-S kinase activity (unpublished observations). The secretagogue action of IpOCOC9 has therefore tentatively, at least partly, been attributed to a direct protein kinase C activation. In the present studies, we examined the influence of IpOCOC9 and DACPME on histamine release triggered by an ensuing exposure to anti-IgE, the calcium ionophore A23187, formyl-methionyl-leucyl-phenylalanine (FMLP), or 4 beta-phorbol 12-myristate 13-acetate (PMA). It is shown that IpOCOC9-treatment of cells results in either enhancement or reduction of the release induced by anti-IgE or by A23187, whereas FMLP-induced release is consistently reduced and PMA-induced release consistently enhanced by such a treatment. Treatment of cells with DACPME enhances but does not reduce anti-IgE-triggered release, whereas FMLP-induced release is not affected. Pretreatment of the cells with other putative protein kinase C activators like PMA, sn-1-oleoyl-2-acetyl-glycerol (OAG), 1,2-dioctanoyl-glycerol (DiC8) or the glycerol derivative sn-1,2-diacetyl-3-decanoyl-glycerol (DiC2OCOC9) affects secretagogue-induced basophil histamine release according to specific patterns similar to but not identical with those recorded for IpOCOC9 and DACPME. Thus, e.g., DiC2OCOC9 consistently reduces but does not enhance anti-IgE-triggered release. These data show that limited structural changes of IpOCOC9 may qualitatively affect its modulating properties in the human basophil histamine release system.

Topics: Calcimycin; Cyclopentanes; Decanoic Acids; Diglycerides; Glyceryl Ethers; Histamine Release; Humans; Immunoglobulin E; Leukocytes; N-Formylmethionine Leucyl-Phenylalanine; Tetradecanoylphorbol Acetate; Triglycerides