ABSTRACT
BACKGROUND: NASH causes a tremendous health care burden in the United States. A glucagon-like peptide-1 agonist, semaglutide (Sema), treatment resulted in hepatic steatosis reduction in clinical trials of NASH. Lysophosphatidic acid receptor 1 antagonists are known to have antifibrotic effects in several organs. We tested Sema and a novel lysophosphatidic acid receptor 1 antagonist, EPGN2154, individually and in combination to evaluate their efficacy for NASH remission in preclinical models. METHODS: In the present study, we used (1) C57Bl6/J wild-type mice fed on a high-fat, high-carbohydrate (HFHC) diet for 16 weeks and (2) leptin-deficient mice (ob/ob) fed on an Amylin liver NASH diet for 16 weeks. After 16 weeks, the mice were randomly distributed in equal numbers in (1) no-drug, (2) EPGN2154, (3) Sema, and (4) EPGN2154+Sema treatment groups for 8 additional weeks at a dosage of 10 mg/kg body weight for EPGN2154 (oral gavage, 5 days a week) and 6.17 µg/kg body weight of Sema (subcutaneous injection every alternate day, 3 days a week). RESULTS: In the wild-type-high-fat, high-carbohydrate model, we observed the most body weight loss in the EPGN2154+Sema combination group compared to the other treatment groups. All groups led to a significant reduction in alanine transaminase levels when compared to high-fat, high-carbohydrate-fed wild type. However, no significant difference in alanine transaminase levels was observed among the treatment groups. In the ob/ob mice study, Sema did not cause body weight loss. Moreover, the EPGN2154 and the combination groups had a lower NAFLD Activity Score and incidence of advanced-stage hepatic fibrosis than the Sema group. CONCLUSIONS: EPGN2154 demonstrated a hepato-protective effect independent of body weight loss in preclinical NASH models.
Subject(s)
Non-alcoholic Fatty Liver Disease , Animals , Mice , Non-alcoholic Fatty Liver Disease/drug therapy , Non-alcoholic Fatty Liver Disease/etiology , Receptors, Lysophosphatidic Acid/therapeutic use , Alanine Transaminase , Body Weight , Diet, High-Fat/adverse effects , Carbohydrates/therapeutic use , Weight LossABSTRACT
Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.
Subject(s)
Blood Platelets/cytology , Cell Differentiation/physiology , Megakaryocytes/cytology , Megakaryocytes/metabolism , Receptors, Lysosphingolipid/metabolism , Animals , Blotting, Southern , Blotting, Western , Bone Marrow Cells/cytology , Cell Differentiation/genetics , Cell Line , Cells, Cultured , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Transmission , Platelet Membrane Glycoprotein IIb/metabolism , Receptors, Lysosphingolipid/genetics , Reverse Transcriptase Polymerase Chain Reaction , Thrombocytopenia/genetics , Thrombocytopenia/metabolism , Thrombocytopenia/pathology , Thrombopoietin/bloodABSTRACT
Mast cells are bone marrow-derived effector cells that can initiate inflammatory responses to infectious organisms or allergens by releasing a multitude of pro-inflammatory factors including prostaglandin (PG) D(2). We demonstrate that primary murine bone marrow-derived mast cells (BMMCs) express the PGD(2) receptor; chemoattractant receptor-homologous molecule expressed on T(h) class 2 cells (CRT(h)2). Activation of CRT(h)2 on BMMC by PGD(2) or the CRT(h)2-specific agonist, 13,14-dihydro-15-keto-prostaglandin D(2) (DK-PGD(2)), resulted in signaling response including Ca(2+) mobilization and phosphorylation of the p42/p44 extracellular signal-regulated kinases (ERKs) kinases. Phosphorylation of the ERKs could be blocked by pertussis toxin, as well as a small molecule antagonist of CRT(h)2, Compound A. Activation of CRT(h)2 on BMMC also resulted in the up-regulation of CD23 and CD30 on the cell surface, as well as CD62L shedding. Finally, PGD(2) and DK-PGD(2) induced the migration of BMMC in vitro and in vivo in response to an intra-dermal DK-PGD(2) injection. Both these processes were inhibited by the CRT(h)2 antagonist. These results raise the possibility that the functional consequences of the PGD(2)-CRT(h)2 interaction on mast cells may be relevant in allergic inflammation.
Subject(s)
Mast Cells/metabolism , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Animals , Bone Marrow/pathology , Calcium/immunology , Calcium/metabolism , Cell Movement/immunology , Cells, Cultured , Hypersensitivity, Delayed/immunology , Ki-1 Antigen/genetics , Ki-1 Antigen/metabolism , L-Selectin/genetics , L-Selectin/metabolism , Mast Cells/immunology , Mast Cells/pathology , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 3/immunology , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Prostaglandin D2/analogs & derivatives , Prostaglandin D2/metabolism , Receptors, IgE/genetics , Receptors, IgE/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Receptors, Prostaglandin/genetics , Receptors, Prostaglandin/immunology , Signal TransductionABSTRACT
A FITC-induced allergic contact hypersensitivity model was used to investigate the role that the prostaglandin D(2) receptor-chemoattractant receptor-homologous molecule expressed on T(h)2 cells (CRTH2) plays in modulating cutaneous inflammation. Our results show that inhibition of CRTH2, achieved via administration of a potent, small molecule antagonist, Compound A (Cmpd A), effectively blocked edema formation and greatly reduced the inflammatory infiltrate and skin pathology observed in drug vehicle-treated animals. Gene expression analysis revealed that Cmpd A administration down-regulated the transcription of a wide range of pro-inflammatory mediators. This correlated with decreases in cytokine and chemokine protein levels, notably IL-4, IL-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, GRO-alpha, MIP-2 and thymic stromal lymphopoietin (TSLP) in FITC-challenged ears. The administration of an anti-TSLP-neutralizing antibody was only partially effective in lowering the FITC-induced inflammatory infiltrate and cytokine production compared with the CRTH2 antagonist. Taken together, these data suggest that blockade of CRTH2 inhibits multiple pathways leading to cutaneous inflammation in this model. This suggests that CRTH2 antagonism may be a viable route for therapeutic intervention in allergic skin diseases, such as atopic dermatitis.
Subject(s)
Dermatitis, Allergic Contact/drug therapy , Prostaglandin Antagonists/therapeutic use , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Animals , Cell Line , Cytokines/immunology , Dermatitis, Allergic Contact/immunology , Female , Fluorescein-5-isothiocyanate/adverse effects , Gene Expression/drug effects , Humans , Mice , Mice, Inbred BALB C , Prostaglandin D2/antagonists & inhibitors , Receptors, Immunologic/immunology , Receptors, Prostaglandin/immunology , Th2 Cells/drug effects , Th2 Cells/immunologyABSTRACT
Prostaglandin D(2) (PGD(2)) and its receptor chemoattractant receptor homologous molecule expressed on T(h)2 cells (CRTH2) have been implicated in the pathogenesis of numerous allergic diseases. We investigated the role of PGD(2) and CRTH2 in allergic cutaneous inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated cutaneous inflammation caused by either repeated epicutaneous ovalbumin or FITC sensitization. Gene expression and ELISA analysis revealed that there was reduced pro-inflammatory cytokine mRNA or protein produced. Importantly, the CRTH2 antagonist reduced total IgE, as well as antigen-specific IgE, IgG1 and IgG2a antibody levels. This reduction in antibody production correlated to reduced cytokines produced by splenocytes following in vitro antigen challenge. An examination of skin CD11c(+) dendritic cells (DC) showed that in mice treated with the CRTH2 antagonist, there was a decrease in the number of these cells that migrated to the draining lymph nodes in response to FITC application to the skin. Additionally, naive CD4(+) T lymphocytes co-cultured with skin-derived DC from CRTH2 antagonist-treated mice showed a reduced ability to produce a number of cytokines compared with DC from vehicle-treated mice. Collectively, these findings suggest that CRTH2 has a pivotal role in mediating the inflammation and the underlying immune response following epicutaneous sensitization.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cytokines/analysis , Dendritic Cells/immunology , Dermatitis, Atopic/immunology , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Animals , Antigens, CD/drug effects , Antigens, CD/metabolism , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , Cytokines/drug effects , Cytokines/immunology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Dermatitis, Atopic/chemically induced , Dermatitis, Atopic/pathology , Female , Gene Expression/drug effects , Gene Expression/immunology , Immunoglobulins/blood , Immunoglobulins/drug effects , Mice , Mice, Inbred BALB C , Ovalbumin/immunology , Prostaglandin Antagonists/pharmacology , Prostaglandin D2/immunology , Prostaglandin D2/metabolism , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Up-RegulationABSTRACT
Idiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-ß pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.
Subject(s)
Idiopathic Pulmonary Fibrosis/genetics , MAP Kinase Kinase Kinases/metabolism , Myofibroblasts/metabolism , Animals , Biopsy , Cell Differentiation , Female , Humans , Idiopathic Pulmonary Fibrosis/metabolism , Lung/pathology , Mice , Mice, SCID , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Signal Transduction , Tomography, X-Ray Computed , Transforming Growth Factor beta/metabolismABSTRACT
The alpha4beta1 integrin (very late antigen-4, VLA-4) plays an important role in the migration of lymphocytes, monocytes, and eosinophils, but not neutrophils, to sites of inflammation. Pharmacological antagonism of VLA-4 is an attractive prospect for the treatment of predominantly eosinophil mediated diseases such as asthma and allergic rhinitis. We report here on a potent and selective, small molecule VLA-4 inhibitor, (2S)-3-(2', 5'-dichlorobiphenyl-4-yl)-2-({[1-(2-methoxybenzoyl)piperidin-3-yl]carbonyl}amino) propanoic acid, compound 1, and characterize the antagonist activities of this molecule in various cell-based assays and in an animal model of eosinophil migration. Compound 1 inhibited VLA-4/ vascular cell adhesion molecule-1(VCAM-1) interactions with in vitro potencies (IC50 value of 210 nM) in VLA-4-expressing Ramos cells, although the compound did not inhibit cell adhesion to fibronectin via alpha5beta1 integrin (very late antigen-5, VLA-5). Blockade of phorbol-12-myristate-13-acetate (PMA)- or Mn2+-stimulated VLA-4 interactions with compound 1 was observed in human T lymphocytes (IC50 value of 230 nM), human eosinophils (IC50 value of 4.0 microM) and mouse eosinophils (IC50 value of 1.6 microM). Furthermore, compound 1 administered by intraperitoneal injection inhibited eosinophil infiltration in a dose-dependent manner by up to 80% in an air pouch model. These data support the use of small molecule VLA-4 antagonists in the treatment of relevant diseases, such as asthma, atopic dermatitis, or allergic rhinitis.
Subject(s)
Anti-Allergic Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Chemotaxis, Leukocyte/drug effects , Eosinophilia/prevention & control , Eosinophils/drug effects , Integrin alpha4beta1/antagonists & inhibitors , Polychlorinated Biphenyls/pharmacology , Skin Diseases/prevention & control , Animals , Anti-Allergic Agents/pharmacokinetics , Anti-Allergic Agents/therapeutic use , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/therapeutic use , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Cell Adhesion/drug effects , Chemokine CCL11 , Chemokines, CC , Disease Models, Animal , Dose-Response Relationship, Drug , Eosinophilia/chemically induced , Eosinophilia/metabolism , Eosinophilia/physiopathology , Eosinophils/metabolism , Female , Fibronectins/metabolism , Humans , Integrin alpha4beta1/metabolism , Integrin alpha5beta1/metabolism , Interleukin-5/biosynthesis , Interleukin-5/genetics , Jurkat Cells , Lymphocyte Activation , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Polychlorinated Biphenyls/pharmacokinetics , Polychlorinated Biphenyls/therapeutic use , Skin Diseases/chemically induced , Skin Diseases/metabolism , Skin Diseases/physiopathology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Tetradecanoylphorbol Acetate , Time Factors , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Involvement of nuclear factor-kappaB (NF-kappaB) in cell survival and proliferation of multiple myeloma has been well established. In this study we observed that NF-kappaB is constitutively activated in all human myeloma cell lines, thus confirming the previous studies. In addition, we found the phosphorylation of p65 subunit of NF-kappaB in addition to the phosphorylation of IkappaBalpha and the activation of NF-kappaB DNA binding and that various target genes of NF-kappaB including bcl-x(L), XIAP, c-IAP1, cyclin D1, and IL-6 are up-regulated. We then examined the effect of a novel IkappaB kinase inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile (ACHP). When myeloma cells were treated with ACHP, the cell growth was efficiently inhibited with IC(50) values ranging from 18 to 35 mumol/L concomitantly with inhibition of the phosphorylation of IkappaBalpha/p65 and NF-kappaB DNA-binding, down-regulation of the NF-kappaB target genes, and induction of apoptosis. In addition, we observed the treatment of ACHP augmented the cytotoxic effects of vincristine and melphalan (l-phenylalanine mustard), conventional antimyeloma drugs. These findings indicate that IkappaB kinase inhibitors such as ACHP can sensitize myeloma cells to the cytotoxic effects of chemotherapeutic agents by blocking the antiapoptotic nature of myeloma cells endowed by the constitutive activation of NF-kappaB.
Subject(s)
I-kappa B Proteins/antagonists & inhibitors , I-kappa B Proteins/pharmacology , Multiple Myeloma/pathology , NF-kappa B/pharmacology , Nicotinic Acids/pharmacology , Nitriles/pharmacology , Cell Proliferation , Cytokines/biosynthesis , DNA/metabolism , Humans , I-kappa B Proteins/metabolism , Phosphorylation , Tumor Cells, Cultured , Up-RegulationABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-ß has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-ß-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-ß stimulation. TGF-ß-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.
Subject(s)
Bleomycin/pharmacology , Lung Injury/chemically induced , Lung Injury/metabolism , MAP Kinase Kinase Kinases/metabolism , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/metabolism , Transforming Growth Factor beta/metabolism , A549 Cells , Animals , Bronchoalveolar Lavage/methods , Cell Line, Tumor , Disease Models, Animal , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , HeLa Cells , Humans , Idiopathic Pulmonary Fibrosis/metabolism , Indoles/pharmacology , Lung/drug effects , Lung/metabolism , Mice , Mice, Inbred C57BL , Pyridones/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung inflammation resulting in a progressive decline in lung function whose principle cause is cigarette smoke. MAP3K19 is a novel kinase expressed predominantly by alveolar and interstitial macrophages and bronchial epithelial cells in the lung. We found that MAP3K19 mRNA was overexpressed in a limited sampling of lung tissue from COPD patients, and a closer examination found it to be overexpressed in bronchoalveolar macrophages from COPD patients, as well as the bronchial epithelium and inflammatory cells in the lamina propria. We further found MAP3K19 to be induced in various cell lines upon environmental stress, such as cigarette smoke, oxidative and osmotic stress. Exogenous expression of MAP3K19 in cells caused an upregulation of transcriptionally active NF-κB, and secretion of the chemokines CXCL-8, CCL-20 and CCL-7. Inhibition of MAP3K19 activity by siRNA or small molecular weight inhibitors caused a decrease in cigarette smoke-induced inflammation in various murine models, which included a decrease in pulmonary neutrophilia and KC levels. In a chronic cigarette smoke model, inhibition of MAP3K19 significantly attenuated emphysematous changes in airway parenchyma. Finally, in a viral exacerbation model, mice exposed to cigarette smoke and influenza A virus showed a decrease in pulmonary neutrophilia, pro-inflammatory cytokines and viral load upon inhibition of MAP3K19. Collectively, these results suggest that inhibition of MAP3K19 may represent a novel strategy to target COPD that promises to have a potential therapeutic benefit for patients.
Subject(s)
Inflammation Mediators/metabolism , MAP Kinase Kinase Kinases/metabolism , Pneumonia/enzymology , Pulmonary Disease, Chronic Obstructive/enzymology , Respiratory System/enzymology , Smoking/adverse effects , Adult , Aged , Aged, 80 and over , Animals , Blotting, Western , Cell Line, Tumor , Cytokines/metabolism , Epithelial Cells/enzymology , Epithelial Cells/metabolism , Female , Gene Expression , HEK293 Cells , Humans , MAP Kinase Kinase Kinases/genetics , Macrophages/enzymology , Male , Mice , Mice, Inbred BALB C , Middle Aged , NF-kappa B/metabolism , Pneumonia/etiology , Pneumonia/genetics , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/metabolism , RNA Interference , Respiratory System/metabolism , Respiratory System/pathology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
1 Pulmonary inflammatory diseases such as asthma are characterized by chronic, cell-mediated inflammation of the bronchial mucosa. 2 Recruitment and activation of inflammatory cells is orchestrated by a variety of mediators such as cytokines, chemokines, or adhesion molecules, the expression of which is regulated via the transcription factor nuclear factor kappa B (NF-kappaB). 3 NF-kappaB signaling is controlled by the inhibitor of kappa B kinase complex (IKK), a critical catalytic subunit of which is IKK-beta. 4 We identified COMPOUND A as a small-molecule, ATP-competitive inhibitor selectively targeting IKK-beta kinase activity with a K(i) value of 2 nM. 5 COMPOUND A inhibited stress-induced NF-kappaB transactivation, chemokine-, cytokine-, and adhesion molecule expression, and T- and B-cell proliferation. 6 COMPOUND A is orally bioavailable and inhibited the release of LPS-induced TNF-alpha in rodents. 7 In mice COMPOUND A inhibited cockroach allergen-induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model of airway inflammation. 8 COMPOUND A was well tolerated by rodents over 3 weeks without affecting weight gain. 9 Furthermore, in mice COMPOUND A suppressed edema formation in response to arachidonic acid, phorbol ester, or edema induced by delayed-type hypersensitivity. 10 These data suggest that IKK-beta inhibitors offer an effective therapeutic approach for inhibiting chronic pulmonary inflammation.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Oxazines/pharmacology , Pneumonia/prevention & control , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/biosynthesis , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Edema/prevention & control , Female , Humans , I-kappa B Kinase , I-kappa B Proteins/metabolism , Leukocytes/cytology , Leukocytes/drug effects , Leukocytes/physiology , Male , Mice , Mice, Inbred BALB C , NF-KappaB Inhibitor alpha , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Oxazines/adverse effects , Oxazines/pharmacokinetics , Phosphorylation , Pneumonia/immunology , Pyridines/adverse effects , Pyridines/pharmacokinetics , Rats , Rats, Wistar , Signal Transduction/drug effects , Transcriptional Activation/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolismABSTRACT
We previously showed that ramatroban (Baynastrade mark), a thromboxane A(2) (TxA(2)) antagonist, had inhibited prostaglandin D(2) (PGD(2))-stimulated human eosinophil migration mediated through activation of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). However, detailed pharmacological characterization of its inhibitory activity has not been described. In the present study, we showed that [(3)H]ramatroban bound to a single receptor site on CRTH2 transfectants with a similar K(d) value (7.2 nM) to a TxA(2) receptor (8.7 nM). We also demonstrated that ramatroban inhibited PGD(2)-, 15-deoxy-Delta(12, 14)-PGJ(2) (15d-PGJ(2))- and indomethacin-induced calcium responses on CRTH2 transfectants in a competitive manner with similar pA(2) values (8.5, 8.5, and 8.6, respectively). This is the first report showing the evidence for direct binding of ramatroban to CRTH2, revealing its competitive inhibitory effects and another interesting finding that PGD(2), indomethacin and 15d-PGJ(2) share the same binding site with ramatroban on CRTH2.
Subject(s)
Carbazoles/pharmacology , Indomethacin/pharmacology , Prostaglandin D2/pharmacology , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Sulfonamides/pharmacology , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Binding, Competitive , Bridged Bicyclo Compounds, Heterocyclic , Calcium/metabolism , Carbazoles/metabolism , Cell Line , Cell Movement/drug effects , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Fatty Acids, Unsaturated , Humans , Hydrazines/pharmacology , Models, Biological , Pentanoic Acids/pharmacology , Prostaglandin D2/analogs & derivatives , Pyridines/pharmacology , Receptors, Immunologic/agonists , Receptors, Immunologic/genetics , Receptors, Prostaglandin/agonists , Receptors, Prostaglandin/genetics , Sulfonamides/metabolism , Transfection , TritiumABSTRACT
The family of phosphoinositide 3-kinases (PI3K) regulates fundamental cellular responses such as proliferation, apoptosis, motility, and adhesion. In particular, the PI3K gamma isoform plays a critical role in the control of cell migration. Despite the attractiveness of PI3-kinases as drug targets, drug discovery efforts have been hampered by the lack of appropriate lipid kinase assay formats suitable for high-throughput screening. The authors report the development of a simple and robust 384-well plate assay that is based on(33) P-phosphate transfer from radiolabeled [gamma(33) P]ATP to phosphatidylinositol immobilized on Maxisorp plates. The established assay format for PI3K gamma was easily adapted to the automated screening platform and was successfully employed for high-throughput screening. Enzymatic and inhibition characteristics of recombinant human PI3K gamma determined with the plate assay are in very good agreement with previously reported values determined in other assay formats. Maximal catalytic activity of PI3K gamma was observed at pH 7.0. The apparent K(m) value for ATP using a 1:1 mixture of phosphatidylinositol and phosphatidylserine was determined to be 7.3 microM (6.0-8.6 microM, 95% confidence interval [CI]). IC(50) values for known PI3-kinase inhibitors were determined to be 1.45 nM (1.17-1.80 nM, 95% CI) for wortmannin and estimated from partial inhibition data to be 1400, 2830, and 21,400 nM for quercetin, LY294002, and staurosporine, respectively. This novel assay approach allows for screening of inhibitors of lipid kinases in high-throughput mode and thereby may facilitate the identification of novel inhibitory structures for drug development.
Subject(s)
Biological Assay/instrumentation , Biological Assay/methods , Enzyme Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phospholipids/metabolism , Adenosine Triphosphate/metabolism , Androstadienes/pharmacology , Class Ib Phosphatidylinositol 3-Kinase , Drug Evaluation, Preclinical/methods , Humans , Isoenzymes/genetics , Lipid Metabolism , Microchemistry/instrumentation , Microchemistry/methods , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositols/metabolism , Phospholipids/chemistry , Phosphorus Radioisotopes/chemistry , Quercetin/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Staurosporine/pharmacology , WortmanninABSTRACT
Prostaglandin D(2), the ligand for the G protein-coupled receptors DP1 and CRTH2, has been implicated in the pathogenesis of the allergic response in diseases such as asthma, rhinitis, and atopic dermatitis. This prostanoid also fulfills a number of physiological, anti-inflammatory roles through its receptor DP1. We investigated the role of PGD(2) and CRTH2 in allergic pulmonary inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated inflammation caused by either acute or subchronic sensitization using the cockroach egg antigen. Gene expression and ELISA analysis revealed that there was reduced proinflammatory cytokine mRNA or protein produced, as well as a wide array of genes associated with the Th2-type proinflammatory response. Importantly, the CRTH2 antagonist reduced antigen-specific IgE, IgG1, and IgG2a antibody levels as well as decreased mucus deposition and leukocyte infiltration in the large airways. Collectively, these findings suggest that the PGD(2)-CRTH2 activation axis has a pivotal role in mediating the inflammation and the underlying immune response in a T cell-driven model of allergic airway inflammation.
Subject(s)
Bronchial Hyperreactivity/complications , Bronchial Hyperreactivity/prevention & control , Down-Regulation/genetics , Pneumonia/complications , Pneumonia/genetics , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Allergens/immunology , Animals , Antibodies/blood , Bronchial Hyperreactivity/blood , Bronchial Hyperreactivity/immunology , Cockroaches , Cytokines/biosynthesis , Disease Models, Animal , Gene Expression Profiling , Humans , Hypersensitivity/complications , Hypersensitivity/immunology , Lung/immunology , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred BALB C , Pneumonia/blood , Pneumonia/immunologyABSTRACT
The cinnabaramides A-G (1-7) were isolated from a terrestrial strain of Streptomyces as potent and selective inhibitors of the human 20S proteasome. Their chemical and biological properties resemble those of salinosporamide A, a recently identified lead compound from an obligate marine actinomycete, which is currently under development as an anticancer agent. Cinnabaramides F and G (6, 7) combine essential structural features of salinosporamide A and lactacystin and show about equal potency in vitro, with IC50 values in the 1 nM range. The properties and phylogenetic position of the producer organism, the production and isolation of compounds 1-7, their structure elucidation by MS and NMR, and their biological activities are reported. Additionally, an X-ray crystal structure was obtained from cinnabaramide A (1).
Subject(s)
Acetylcysteine/analogs & derivatives , Lactones , Proteasome Inhibitors , Pyrroles , Streptomyces/chemistry , Acetylcysteine/blood , Acetylcysteine/chemistry , Acetylcysteine/isolation & purification , Acetylcysteine/pharmacology , Crystallography, X-Ray , Humans , Lactones/blood , Lactones/isolation & purification , Lactones/pharmacology , Molecular Conformation , Molecular Structure , Pyrroles/blood , Pyrroles/isolation & purification , Pyrroles/pharmacologyABSTRACT
BACKGROUND: To investigate the functional role of protease activated receptor (PAR) -2 in T lymphocytes, we analyzed TCR-mediated inflammatory cytokine production using PAR-2 deficient (KO) and wild type (WT) mice. METHODS: Production of serum IgE and cytokines by spleen CD4+ T cells was determined in OVA-sensitized and OVA-challenged mice of PAR-2 KO in contrast to WT mice. Phosphorylation of JNK1 and 2 was determined by Western blotting. RESULTS: A reduction in serum levels of IgE and IL-4 production by splenic CD4+ T cells from OVA-sensitized and OVA-challenged KO mice compared to WT mice was observed. By contrast, IFN-gamma production was upregulated after antigen stimulation in KO mice. Anti-CD3-mediated phosphorylation of JNK1 was upregulated in splenic CD4+ T cells from KO mice compared to WT mice. CONCLUSIONS: PAR-2 participates in the regulation of T cell cytokine production that may be caused by modulation of JNK1 phosphorylation.
Subject(s)
Antigens/immunology , CD4-Positive T-Lymphocytes/metabolism , Down-Regulation/genetics , Interferon-gamma/biosynthesis , Interleukin-4/antagonists & inhibitors , Receptor, PAR-2/deficiency , Up-Regulation/genetics , Animals , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , Down-Regulation/immunology , Interferon-gamma/metabolism , Interleukin-4/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 8/metabolism , Ovalbumin/immunology , Phosphorylation , Receptor, PAR-2/genetics , Up-Regulation/immunologyABSTRACT
The discovery of a second receptor for prostaglandin D2, chemoattractant receptor-homologous molecule expressed on T helper type 2 (CRTH2), has revived significant efforts into the development of small-molecule antagonists for allergic diseases as the receptor is predominantly expressed on cells such as eosinophils, TH2 cells and basophils, which are major pro-inflammatory cells in diseases such as asthma and allergic rhinitis. This brief review serves to illustrate current patent literature focusing on the similarities and differences of an ever-growing number of CRTH2 antagonists emerging from corporate laboratories.
Subject(s)
Hypersensitivity/drug therapy , Hypersensitivity/pathology , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/physiology , Prostaglandin Antagonists/therapeutic use , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/physiology , Receptors, Prostaglandin/antagonists & inhibitors , Receptors, Prostaglandin/physiology , Animals , Humans , Hypersensitivity/immunology , Prostaglandin Antagonists/pharmacologyABSTRACT
Zymosan and carrageenan represent two inflammatory stimuli leading to significant neutrophilia when injected into mice. Despite several similarities between the two proinflammatory agents, the mechanisms leading to neutrophil influx into the site of stimulus injection are unclear. As demonstrated by antibody (Ab) studies directed against adhesion molecules, L-selectin was pivotal for zymosan-induced but not carrageenan-induced pleurisy. Zymosan but not carrageenan injection into the pleural cavity caused blood neutrophilia and significant release of neutrophils from the bone marrow, events that were inhibited by anti-L-selectin but not anti-Mac-1 Ab pretreatment. Pertussis toxin, known to regulate cell efflux, abrogated both zymosan- and carrageenan-induced pleurisy, but only zymosan-induced neutrophil release from the bone marrow. Dexamethasone, known to inhibit pleurisy induced by either stimulus, had no effect on bone marrow neutrophil numbers. The G(i/o) G protein-coupled H4 histamine receptor is highly expressed in the bone marrow and on leukocytes and plays an important role in zymosan-induced pleurisy in vivo. Zymosan-triggered neutrophil release from bone marrow was abrogated by pretreatment of mice with thioperamide, a known H(3/4) receptor antagonist, whereas H1 and H2 receptor antagonists had no effect. Moreover, histamine itself, when injected intravenously, led to a similar time- and dose-dependent decrease of neutrophil numbers in the bone marrow that was inhibited by thioperamide. Because the H3 receptor is not expressed on neutrophils, these findings indicate that both H4 and L-selectin regulate zymosan-induced neutrophil release from bone marrow and subsequent infiltration in the pleurisy model.
Subject(s)
Carrageenan/pharmacology , L-Selectin/physiology , Neutrophil Infiltration/drug effects , Receptors, G-Protein-Coupled/physiology , Receptors, Histamine/physiology , Zymosan/pharmacology , Animals , Bone Marrow , Female , Mice , Mice, Inbred BALB C , Neutrophil Infiltration/physiology , Receptors, Histamine H4ABSTRACT
The recently identified histamine receptor, H4, was shown to be primarily expressed on leukocytes and has been implicated in the activation of lymphocytes, eosinophils, and mast cells in vitro. Its function in vivo, however, has not yet been characterized. We present evidence for a critical role of H4 receptor in the mast cell-dependent recruitment of neutrophils. Mice injected with zymosan into the pleural cavity developed massive neutrophilia within hours after challenge. Neutrophilia was dose-dependently reduced when mice were pretreated with thioperamide, a known H(3/4) receptor antagonist, whereas H1 and H2 receptor antagonists lacked efficacy. Similarly, a 70 to 80% reduction in neutrophils in the pleural cavity compared with wild-type animals was noted in mice lacking mast cells (W/W(v) mice); mice deficient in MyD88 (MyD88(-/-)); a critical component of the signaling cascade of the major receptor for zymosan, toll-like receptor 2 (TLR2); or in mice pretreated with a functionally antagonistic anti-TLR2 antibody. The residual 20% neutrophil infiltration seen in mast cell-deficient and MyD88(-/-) mice was not further reduced by thioperamide. Neutrophilia was completely restored by transferring wild-type bone marrow-derived mast cells into MyD88(-/-) or W/W(v) mice. Interestingly, when neutrophilia was evoked by carrageenan injection, mast cell depletion and thioperamide had no effect. Various inflammatory mediators were detectable in the pleural cavity of zymosan-challenged mice. Upon pretreatment with thioperamide, reduced levels of the neutrophil chemoattractant leukotriene B4 were observed, providing a mechanistic explanation for the prevention of neutrophilia by H4 receptor antagonism.
Subject(s)
Leukotriene B4/biosynthesis , Mast Cells/metabolism , Neutrophil Infiltration/drug effects , Pleurisy/pathology , Receptors, G-Protein-Coupled , Receptors, Histamine/physiology , Animals , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Carrageenan , Cimetidine/pharmacology , Cytokines/metabolism , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Histamine Antagonists/pharmacology , Histamine H1 Antagonists/pharmacology , Histamine H2 Antagonists/pharmacology , Histamine Release/drug effects , Mast Cells/drug effects , Mice , Mice, Inbred BALB C , Piperidines/pharmacology , Pleurisy/chemically induced , Pyrilamine/pharmacology , Receptors, Histamine/drug effects , Receptors, Histamine H3/drug effects , Receptors, Histamine H3/physiology , Receptors, Histamine H4 , ZymosanABSTRACT
Contact hypersensitivity (CHS) induced by a hapten is thought to be mediated by T helper type 1 (Th1) cells. However, FITC can induce contact allergy in vivo, and in vitro studies suggest that this response is Th2-type driven. We compared CHS reactions induced by FITC or dinitrofluorobenzene (DNFB), a well-known Th1 inducing hapten, in Balb/c mice, C57/B6 mice, and several gene knock-out mice, and investigated the role of Th1/Th2 cytokines, T cell populations, eosinophils, and mast cells. Balb/c mice (Th2 dominant strain) had a stronger response to FITC than C57/B6 mice (Th1 dominant strain). The skin inflammation was characterized by edema and eosinophilia, and serum IgE levels were elevated following FITC challenge. All responses were enhanced by a second round of sensitization. Anti-TNF-alpha or anti-very late antigen-4 (VLA-4) antibody partly inhibited both FITC- and DNFB-induced CHS. Pretreatment of mice with anti-IL-4 antibody, anti-IL-5 antibody, recombinant INF-gamma, or the mast-cell depleting agent 48/80 significantly diminished edema formation, and Stat6(-/-) mice were fully protected from FITC-induced CHS, while DNFB-induced CHS was enhanced (Stat6(-/-), mast cell depletion) or not affected (anti-IL-5 antibody). Further, mice lacking CD4(+) T cells and mice lacking both CD8 and MHC II showed very little reaction at all to FITC, while the absence of CD8 T cells alone or MHC II alone conferred partial protection only. These findings indicate a contribution of MHC II-independent CD4(+) T cells and/or CD4(+) NKT cells to the Th2 response triggered by FITC in vivo, and makes FITC-induced CHS a suitable animal model for atopic dermatitis.