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1.
Pulm Pharmacol Ther ; 38: 17-26, 2016 06.
Article in English | MEDLINE | ID: mdl-27133900

ABSTRACT

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1ß upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1ß in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Dexamethasone/pharmacology , Mucin 5AC/genetics , Pregnadienediols/pharmacology , A549 Cells , Anti-Inflammatory Agents/administration & dosage , Bronchi/cytology , Bronchi/drug effects , Cell Line , Dexamethasone/administration & dosage , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Glucocorticoids/administration & dosage , Glucocorticoids/pharmacology , Humans , Inflammation Mediators/metabolism , Interleukin-1beta/metabolism , Mucins/antagonists & inhibitors , Mucins/metabolism , Pregnadienediols/administration & dosage , RNA, Messenger/metabolism , Time Factors
2.
Am J Respir Cell Mol Biol ; 53(1): 22-32, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25692303

ABSTRACT

Alterations in epithelial secretions and mucociliary clearance contribute to chronic bacterial infection in cystic fibrosis (CF) lung disease, but whether CF lungs are unchanged in the absence of infection remains controversial. A proteomic comparison of airway secretions from subjects with CF and control subjects shows alterations in key biological processes, including immune response and proteolytic activity, but it is unclear if these are due to mutant CF transmembrane conductance regulator (CFTR) and/or chronic infection. We hypothesized that the CF lung apical secretome is altered under constitutive conditions in the absence of inflammatory cells and pathogens. To test this, we performed quantitative proteomics of in vitro apical secretions from air-liquid interface cultures of three life-extended CF (ΔF508/ΔF508) and three non-CF human bronchial epithelial cells after labeling of CF cells by stable isotope labeling with amino acids in cell culture. Mass spectrometry analysis identified and quantitated 666 proteins across samples, of which 70 exhibited differential enrichment or depletion in CF secretions (±1.5-fold change; P < 0.05). The key molecular functions were innate immunity (24%), cytoskeleton/extracellular matrix organization (24%), and protease/antiprotease activity (17%). Oxidative proteins and classical complement pathway proteins that are altered in CF secretions in vivo were not altered in vitro. Specific differentially increased proteins-MUC5AC and MUC5B mucins, fibronectin, and matrix metalloproteinase-9-were validated by antibody-based assays. Overall, the in vitro CF secretome data are indicative of a constitutive airway epithelium with altered innate immunity, suggesting that downstream consequences of mutant CFTR set the stage for chronic inflammation and infection in CF airways.


Subject(s)
Bronchi/metabolism , Cystic Fibrosis/metabolism , Proteome/metabolism , Proteomics , Respiratory Mucosa/metabolism , Bronchi/pathology , Cell Line , Chronic Disease , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Proteome/genetics , Respiratory Mucosa/pathology
3.
Am J Physiol Lung Cell Mol Physiol ; 306(8): L797-807, 2014 Apr 15.
Article in English | MEDLINE | ID: mdl-24487386

ABSTRACT

Chronic airway diseases are characterized by inflammation and mucus overproduction. The MUC5AC mucin gene is upregulated by the proinflammatory cytokine interleukin-1 ß (IL-1ß) via activation of cAMP response element-binding protein (CREB) in the NCI-H292 cancer cell line and nuclear factor-κB (NF-κB) in the HBE1 transformed cell line, with each transcription factor binding to a cognate cis site in the proximal or distal region, respectively, of the MUC5AC promoter. We utilized primary differentiated human bronchial epithelial (HBE) and A549 lung adenocarcinoma cells to further investigate the contributions of CREB and NF-κB subunits to the IL-1ß-induced upregulation of MUC5AC. Data show that ligand binding of IL-1ß to the IL-1ß receptor is required to increase MUC5AC mRNA abundance. Chromatin immunoprecipitation analyses show direct binding of CREB to the previously identified cAMP response element site and binding of p65 and p50 subunits to a novel NF-κB site in a mucin-regulatory domain in the proximal promoter and to a previously identified NF-κB site in the distal promoter. P50 binds to both NF-κB sites at 1 h following IL-1ß exposure, but is replaced at 2 h by p65 in A549 cells and by a p50/p65 heterodimer in HBE cells. Thus IL-1ß activates multiple domains in the MUC5AC promoter but exhibits some cell-specific responses, highlighting the complexity of MUC5AC transcriptional regulation. Data show that dexamethasone, a glucocorticoid that transcriptionally represses MUC5AC gene expression under constitutive conditions, also represses IL-1ß-mediated upregulation of MUC5AC gene expression. A further understanding of mechanisms mediating MUC5AC regulation should lead to a honing of therapeutic approaches for the treatment of mucus overproduction in inflammatory lung diseases.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Dexamethasone/pharmacology , Gene Expression Regulation , Interleukin-1beta/pharmacology , Lung Neoplasms/genetics , Mucin 5AC/genetics , NF-kappa B/metabolism , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Anti-Inflammatory Agents/pharmacology , Bronchi/drug effects , Bronchi/metabolism , Cells, Cultured , Chromatin Immunoprecipitation , Cyclic AMP Response Element-Binding Protein/genetics , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Electrophoretic Mobility Shift Assay , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Mucin 5AC/metabolism , NF-kappa B/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/metabolism , Reverse Transcriptase Polymerase Chain Reaction
4.
PLoS One ; 7(7): e41549, 2012.
Article in English | MEDLINE | ID: mdl-22844498

ABSTRACT

PURPOSE: End-organ apoptosis is well-described in progressive sepsis and Multiple Organ Dysfunction Syndrome (MODS), especially where platelets accumulate (e.g. spleen and lung). We previously reported an acute sepsis-induced cytotoxic platelet phenotype expressing serine protease granzyme B. We now aim to define the site(s) of and mechanism(s) by which platelet granzyme B induces end-organ apoptosis in sepsis. METHODS: End-organ apoptosis in murine sepsis (i.e. polymicrobial peritonitis) was analyzed by immunohistochemistry. Platelet cytotoxicity was measured by flow cytometry following 90 minute ex vivo co-incubation with healthy murine splenocytes. Sepsis progression was measured via validated preclinical murine sepsis score. MEASUREMENTS AND MAIN RESULTS: There was evident apoptosis in spleen, lung, and kidney sections from septic wild type mice. In contrast, there was a lack of TUNEL staining in spleens and lungs from septic granzyme B null mice and these mice survived longer following induction of sepsis than wild type mice. In co-incubation experiments, physical separation of septic platelets from splenocytes by a semi-permeable membrane reduced splenocyte apoptosis to a rate indistinguishable from negative controls. Chemical separation by the platelet GPIIb/IIIa receptor inhibitor eptifibatide decreased apoptosis by 66.6±10.6% (p = 0.008). Mice treated with eptifibatide in vivo survived longer following induction of sepsis than vehicle control mice. CONCLUSIONS: In sepsis, platelet granzyme B-mediated apoptosis occurs in spleen and lung, and absence of granzyme B slows sepsis progression. This process proceeds in a contact-dependent manner that is inhibited ex vivo and in vivo by the platelet GPIIb/IIIa receptor inhibitor eptifibatide. The GPIIb/IIIa inhibitors and other classes of anti-platelet drugs may be protective in sepsis.


Subject(s)
Apoptosis/drug effects , Blood Platelets/drug effects , Blood Platelets/pathology , Peptides/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Sepsis/blood , Sepsis/pathology , Animals , Caspases/metabolism , Disease Progression , Eptifibatide , Granzymes/metabolism , Lung/drug effects , Lung/immunology , Lymphocytes/drug effects , Male , Mice , Peptides/therapeutic use , Perforin/metabolism , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Sepsis/drug therapy , Sepsis/metabolism , Spleen/drug effects , Spleen/immunology
5.
J Pharmacol Exp Ther ; 343(1): 225-32, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22743576

ABSTRACT

Glucocorticoids are standard of care for many inflammatory conditions, but chronic use is associated with a broad array of side effects. This has led to a search for dissociative glucocorticoids--drugs able to retain or improve efficacy associated with transrepression [nuclear factor-κB (NF-κB) inhibition] but with the loss of side effects associated with transactivation (receptor-mediated transcriptional activation through glucocorticoid response element gene promoter elements). We investigated a glucocorticoid derivative with a Δ-9,11 modification as a dissociative steroid. The Δ-9,11 analog showed potent inhibition of tumor necrosis factor-α-induced NF-κB signaling in cell reporter assays, and this transrepression activity was blocked by 17ß-hydroxy-11ß-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU-486), showing the requirement for the glucocorticoid receptor (GR). The Δ-9,11 analog induced the nuclear translocation of GR but showed the loss of transactivation as assayed by GR-luciferase constructs as well as mRNA profiles of treated cells. The Δ-9,11 analog was tested for efficacy and side effects in two mouse models of muscular dystrophy: mdx (dystrophin deficiency), and SJL (dysferlin deficiency). Daily oral delivery of the Δ-9,11 analog showed a reduction of muscle inflammation and improvements in multiple muscle function assays yet no reductions in body weight or spleen size, suggesting the loss of key side effects. Our data demonstrate that a Δ-9,11 analog dissociates the GR-mediated transcriptional activities from anti-inflammatory activities. Accordingly, Δ-9,11 analogs may hold promise as a source of safer therapeutic agents for chronic inflammatory disorders.


Subject(s)
Dronabinol/analogs & derivatives , Glucocorticoids/adverse effects , Glucocorticoids/pharmacology , NF-kappa B/antagonists & inhibitors , Response Elements/drug effects , Animals , Dose-Response Relationship, Drug , Dronabinol/chemistry , Dronabinol/pharmacology , Female , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Mice, Knockout , NF-kappa B/metabolism , Response Elements/physiology , Spleen/drug effects , Spleen/metabolism , Treatment Outcome
6.
Nat Med ; 15(7): 808-13, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19525962

ABSTRACT

Self-renewing cell populations such as hematopoietic stem cells and memory B and T lymphocytes might be regulated by shared signaling pathways. The Wnt-beta-catenin pathway is an evolutionarily conserved pathway that promotes hematopoietic stem cell self-renewal and multipotency by limiting stem cell proliferation and differentiation, but its role in the generation and maintenance of memory T cells is unknown. We found that induction of Wnt-beta-catenin signaling by inhibitors of glycogen sythase kinase-3beta or the Wnt protein family member Wnt3a arrested CD8(+) T cell development into effector cells. By blocking T cell differentiation, Wnt signaling promoted the generation of CD44(low)CD62L(high)Sca-1(high)CD122(high)Bcl-2(high) self-renewing multipotent CD8(+) memory stem cells with proliferative and antitumor capacities exceeding those of central and effector memory T cell subsets. These findings reveal a key role for Wnt signaling in the maintenance of 'stemness' in mature memory CD8(+) T cells and have major implications for the design of new vaccination strategies and adoptive immunotherapies.


Subject(s)
CD8-Positive T-Lymphocytes/physiology , Hematopoietic Stem Cells/physiology , Immunologic Memory , Signal Transduction/physiology , Wnt Proteins/physiology , Animals , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta , Hepatocyte Nuclear Factor 1-alpha , Hyaluronan Receptors/analysis , L-Selectin/analysis , Lymphocyte Activation , Mice , Mice, Inbred C57BL , T Cell Transcription Factor 1/physiology , beta Catenin/physiology
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