Michael Schuliga

NF-kappaB Signaling in Chronic Inflammatory Airway Disease

Asthma and chronic obstructive pulmonary disease (COPD) are obstructive airway disorders which differ in their underlying causes and phenotypes but overlap in patterns of pharmacological treatments. In both asthma and COPD, oxidative stress contributes to airway inflammation by inducing inflammatory gene expression. The redox-sensitive transcription factor, nuclear factor (NF)-kappaB (NF-κB), is an important participant in a broad spectrum of inflammatory networks that regulate cytokine activity in airway pathology. The anti-inflammatory actions of glucocorticoids (GCs), a mainstay treatment for asthma, involve inhibition of NF-κB induced gene transcription. Ligand bound GC receptors (GRs) bind NF-κB to suppress the transcription of NF-κB responsive genes (i.e., transrepression). However, in severe asthma and COPD, the transrepression of NF-κB by GCs is negated as a consequence of post-translational changes to GR and histones involved in chromatin remodeling. Therapeutics which target NF-κB activation, including inhibitors of IκB kinases (IKKs) are potential treatments for asthma and COPD. Furthermore, reversing GR/histone acetylation shows promise as a strategy to treat steroid refractory airway disease by augmenting NF-κB transrepression. This review examines NF-κB signaling in airway inflammation and its potential as target for treatment of asthma and COPD.

Annexin-1 signals mitogen-stimulated breast tumor cell proliferation by activation of the formyl peptide receptors (FPRs) 1 and 2

The role of the calcium‐ and phospho‐lipid‐binding protein annexin I (ANXA1) in cell cycle regulation has been investigated in estrogen receptor (ER)‐positive MCF‐7 and ER‐negative MDA‐MB‐231 breast tumor cell lines. In MCF‐7 cells, ANXA1‐targeting small interfering RNA (siRNA) reduced ANXA1 mRNA and protein levels and attenuated cell proliferation induced by FCS, estradiol, or epidermal growth factor. Well‐characterized agonists for the known ANXA1 receptor, FPR2, including the ANXA1 N‐terminal proteolytic product ANXA12_26, lipoxin A4 (LXA4), and the synthetic peptide, Trp‐Lys‐Tyr‐Met‐Val‐D‐Met (WKYMVm), stimulated proliferation of MCF‐7 and MDA‐MB‐231 cells that was attenuated by incubation with FPR2 antagonists WRW4 (1 µM) or Boc2 (100 nM) or by siRNA against FPR2. FCS‐induced mitogenic responses were attenuated by each of the FPR antagonists and by siRNAagainst FPR2 and, to a lesser extent, FPR1. LXA4 increased phosphorylation of Akt, p70S6K but not ERK1/2. Increases in cyclin D1 protein induced by FCS or LXA4 were blocked by the PI3 kinase inhibitor, LY294002, and attenuated by FPR2 antagonism using Boc2. In invasive breast cancer, immunohis‐tochemistry revealed the presence of ANXA1 and its receptor, FPR2, in both tumor epithelium and stromal cells. These observations suggest a novel signaling role for ANXA1 in mitogen‐activated proliferation of breast tumor epithelial cells that is mediated via activation of FPR1 and FPR2.—Khau, T., Langenbach, S. Y., Schu‐liga, M., Harris, T., Johnstone, C. N., Anderson, R. L., Stewart, A. G. Annexin‐1 signals mitogen‐stimulated breast tumor cell proliferation by activation of the formyl peptide receptors (FPRs) 1 and 2. FASEB J. 25, 483_496 (2011). www.fasebj.org

Aquaporin-1 in the choroid plexuses of developing mammalian brain

The normal brain develops within a well-controlled stable internal “milieu” protected by specialised mechanisms referred to collectively as blood–brain barriers. A fundamental feature of this environment is the control of water flow in and out of the developing brain. Because of limited vascularisation of the immature brain, choroid plexuses, via the cerebrospinal fluid, have been proposed as the main route of fluid exchange between the blood and brain interfaces. We describe the temporal expression and appearance of aquaporin-1 (AQP1) which is important for water transfer across adult choroid plexuses. AQP1 expression was studied in rat embryos using real time reverse transcription/polymerase chain reaction. mRNA for AQP1 was present in rat brain at embryonic day 12 (E12) one day before the protein was detectable in the fourth ventricular choroid plexus (the first plexus to appear); its relative levels increased at E13-E14 when more AQP1-immunoreactive cells appeared in all plexuses. The presence of AQP1 was determined immunocytochemically in five different mammalian species (rat, mouse, human, sheep and opossum) in all four choroid plexuses from their earliest appearance. In all five species studied, the appearance of AQP1 immunoreactivity followed the same developmental sequence: the fourth, lateral and, finally, third ventricular choroid plexus. The stage of choroid plexus development when AQP1 was first detected in all five species and in all four choroid plexuses corresponded to the transition between Stages I and II. The cellular localisation of AQP1 in all choroid plexuses, as soon as it was detectable, had the characteristic apical membrane distribution previously described in the adult; a basolateral membrane localisation was also observed.

Tissue and matrix influences on airway smooth muscle function

Asthma is characterized by structural changes in the airways - airway remodelling. These changes include an increase in the bulk of the airway smooth muscle (ASM) and alterations in the profile of extracellular matrix (ECM) proteins in the airway wall. The mechanisms leading to airway remodelling are not well understood. ASM cells have the potential to play a key role in these processes through the production and release of ECM proteins. The ASM cells and ECM proteins are each able to influence the behaviour and characteristics of the other. The modified ECM profile in the asthmatic airway may contribute to the altered behaviour of the ASM cells, such responses to ECM proteins are modulated through the cell surface expression of integrin receptors. ASM cells from asthmatic individuals express different levels of some integrin subunits compared to nonasthmatic ASM cells, which have the potential to further influence their responses to the ECM proteins in the airways. ECM homeostasis requires the presence and activation of matrix metalloproteinases and their tissue inhibitors, which in turn modulate the interaction of the ASM cells and the ECM proteins. Furthermore, the complex interactions of the ASM cells and the ECM in the asthmatic airways and the role played by external stimuli, such as viral infections, to modulate airway remodelling are currently unknown. This review summarises our current understanding of the influence of the ECM on ASM function.

2-Methoxyestradiol Is an Estrogen Receptor Agonist That Supports Tumor Growth in Murine Xenograft Models of Breast Cancer

Purpose: 2-Methoxyestradiol (2MEO) is being developed as a novel antitumor agent based on its antiangiogenic activity, tumor cell cytotoxicity, and apparent lack of toxicity. However, pharmacologic concentrations of 2MEO bind to estrogen receptors (ER). We have therefore examined the ER activity of 2MEO. Experimental Design: Estrogenic actions of 2MEO were evaluated by changes in gene expression of the ER-positive (MCF7) breast tumor cell line and, in vivo, estrogenicity was assessed in breast tumor xenograft models and by measuring endocrine responses in uterus and liver. Results: In the ER-positive breast tumor cell line (MCF7), microarray experiments revealed that 269 of 279 changes in gene expression common to 2MEO and estradiol were prevented by the ER antagonist, ICI 182,780. Changes in the expression of selected genes and their sensitivity to inhibition by ICI 182,780 were confirmed by quantitative reverse transcription–PCR measurement. Activation of ER in MCF7 cells by 2MEO was further confirmed by stimulation of an estrogen response element–dependent reporter gene that was blocked by ICI 182,780 (1 μmol/L). Doses of 2MEO (15-150 mg/kg) that had no antitumor efficacy in either nu/nu BALB/c or severe combined immunodeficient mice bearing ER-negative MDA-MB-435 tumors had uterotropic and hepatic estrogen-like actions. In female nu/nu BALB/c mice inoculated with the estrogen-dependent MCF7 tumor cells, 2MEO (50 mg/kg/d) supported tumor growth. Conclusions: Tumor growth enhancement by 2MEO at doses generating serum levels (100-500 nmol/L) that have estrogenic activity suggests that a conservative approach to the further clinical evaluation of this agent should be adopted and that its evaluation in breast cancer is inappropriate.

Collagen impairs glucocorticoid actions in airway smooth muscle through integrin signalling

Airway wall remodelling in asthma is characterised by a number of structural changes, including an increase in the volume of airway smooth muscle (ASM), and the abundance of the extracellular matrix (ECM) protein, collagen, is increased. We have investigated the mechanism of collagen‐induced glucocorticoid resistance of proliferation, and migration of ASM.

The Inflammatory Actions of Coagulant and Fibrinolytic Proteases in Disease

Aside from their role in hemostasis, coagulant and fibrinolytic proteases are important mediators of inflammation in diseases such as asthma, atherosclerosis, rheumatoid arthritis, and cancer. The blood circulating zymogens of these proteases enter damaged tissue as a consequence of vascular leak or rupture to become activated and contribute to extravascular coagulation or fibrinolysis. The coagulants, factor Xa (FXa), factor VIIa (FVIIa), tissue factor, and thrombin, also evoke cell-mediated actions on structural cells (e.g., fibroblasts and smooth muscle cells) or inflammatory cells (e.g., macrophages) via the proteolytic activation of protease-activated receptors (PARs). Plasmin, the principle enzymatic mediator of fibrinolysis, also forms toll-like receptor-4 (TLR-4) activating fibrin degradation products (FDPs) and can release latent-matrix bound growth factors such as transforming growth factor-β (TGF-β). Furthermore, the proteases that convert plasminogen into plasmin (e.g., urokinase plasminogen activator) evoke plasmin-independent proinflammatory actions involving coreceptor activation. Selectively targeting the receptor-mediated actions of hemostatic proteases is a strategy that may be used to treat inflammatory disease without the bleeding complications of conventional anticoagulant therapies. The mechanisms by which proteases of the coagulant and fibrinolytic systems contribute to extravascular inflammation in disease will be considered in this review.

Transforming growth factor‐β impairs glucocorticoid activity in the A549 lung adenocarcinoma cell line

BACKGROUND AND PURPOSE The lung adenocarcinoma cell line, A549, undergoes epithelial‐mesenchymal cell transition (EMT) in response to TGF‐β. Glucocorticoids do not prevent the EMT response, but TGF‐β induced resistance to the cytokine‐regulatory action of glucocorticoids. We sought to characterize the impairment of glucocorticoid response in A549 cells.

Stimulus-dependent glucocorticoid-resistance of GM-CSF production in human cultured airway smooth muscle.

1 For a subpopulation of asthmatics, symptoms persist even with high doses of glucocorticoids. Glucocorticoids reduce the levels of the proinflammatory and fibrogenic cytokine, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) produced by human cultured airway smooth muscle (ASM). We have contrasted the effects of a synthetic glucocorticoid, dexamethasone, on thrombin‐ and IL‐1α‐stimulated GM‐CSF production in human ASM cells. 2 Although IL‐1α stimulated three‐fold higher levels of GM‐CSF mRNA and protein compared to thrombin, dexamethasone concentration‐dependently reduced IL‐1α‐stimulated GM‐CSF more potently and to a greater extent than the response to thrombin. This pattern of glucocorticoid regulation was also observed at the GM‐CSF mRNA level and was reproduced with other glucocorticoids such as fluticasone propionate. 3 IL‐1α and thrombin stimulated NF‐κB‐dependent luciferase expression equally. Dexamethasone treatment reduced luciferase expression stimulated by both IL‐1α and thrombin. 4 The GM‐CSF mRNA half life was markedly prolonged by IL‐1α compared to thrombin. This IL‐1α‐induced GM‐CSF mRNA stability was prevented by either dexamethasone or the p38MAPK inhibitor, SB203580, neither of which influenced GM‐CSF mRNA stability in thrombin‐treated cells. Dexamethasone inhibited p38MAPK phosphorylation in IL‐1α‐stimulated ASM, whereas thrombin does not stimulate p38MAPK phosphorylation. 5 These data suggest that the mechanism underlying the greater potency and efficacy of glucocorticoids in reducing GM‐CSF synthesis stimulated by IL‐1α depends on inhibition of the involvement of p38MAPK‐induced increases in GM‐CSF message stability.

The plasminogen activation system: new targets in lung inflammation and remodeling

The plasminogen activation system (PAS) and the plasmin it forms have dual roles in chronic respiratory diseases including asthma, chronic obstructive pulmonary disease and interstitial lung disease. Whilst plasmin-mediated airspace fibrinolysis is beneficial, interstitial plasmin contributes to lung dysfunction because of its pro-inflammatory and tissue remodeling activities. Recent studies highlight the potential of fibrinolytic agents, including small molecule inhibitors of plasminogen activator inhibitor-1 (PAI-1), as treatments for chronic respiratory disease. Current data also suggest that interstitial urokinase plasminogen activator is an important mediator of lung inflammation and remodeling. However, further preclinical characterization of uPA as a drug target for lung disease is required. Here we review the concept of selectively targeting the contributions of PAS to treat chronic respiratory disease.