Plasminogen-stimulated airway smooth muscle cell proliferation is mediated by urokinase and annexin A2, involving plasmin-activated cell signalling.

BACKGROUND AND PURPOSE: The conversion of plasminogen into plasmin by interstitial urokinase plasminogen activator (uPA) is potentially important in asthma pathophysiology. In this study, the effect of uPA-mediated plasminogen activation on airway smooth muscle (ASM) cell proliferation was investigated. EXPERIMENTAL APPROACH: Human ASM cells were incubated with plasminogen (0.5-50 µg·mL(-1) ) or plasmin (0.5-50 mU·mL(-1) ) in the presence of pharmacological inhibitors, including UK122, an inhibitor of uPA. Proliferation was assessed by increases in cell number or MTT reduction after 48 h incubation with plasmin(ogen), and by earlier increases in [(3) H]-thymidine incorporation and cyclin D1 expression. KEY RESULTS: Plasminogen (5 µg·mL(-1) )-stimulated increases in cell proliferation were attenuated by UK122 (10 µM) or by transfection with uPA gene-specific siRNA. Exogenous plasmin (5 mU·mL(-1) ) also stimulated increases in cell proliferation. Inhibition of plasmin-stimulated ERK1/2 or PI3K/Akt signalling attenuated plasmin-stimulated increases in ASM proliferation. Furthermore, pharmacological inhibition of cell signalling mediated by the EGF receptor, a receptor trans-activated by plasmin, also reduced plasmin(ogen)-stimulated cell proliferation. Knock down of annexin A2, which has dual roles in both plasminogen activation and plasmin-signal transduction, also attenuated ASM cell proliferation following incubation with either plasminogen or plasmin. CONCLUSIONS AND IMPLICATIONS: Plasminogen stimulates ASM cell proliferation in a manner mediated by uPA and involving multiple signalling pathways downstream of plasmin. Targeting mediators of plasminogen-evoked ASM responses, such as uPA or annexin A2, may be useful in the treatment of asthma.

Transforming growth factor-ß 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-ß. Glucocorticoids do not prevent the EMT response, but TGF-ß induced resistance to the cytokine-regulatory action of glucocorticoids. We sought to characterize the impairment of glucocorticoid response in A549 cells. EXPERIMENTAL APPROACH: A549 cells were exposed to TGF-ß for up to 96 h before glucocorticoid treatment and challenge with IL-1α to assess glucocorticoid regulation of IL-6 and CXCL8 production. Nuclear localization of the glucocorticoid receptor α (GRα) was ascertained by immunofluorescence and Western blotting. Transactivation of the glucocorticoid response element (GRE) was measured with a transfected GRE-secreted human placental alkaline phosphatase reporter. KEY RESULTS: TGF-ß (40-400 pM) reduced the maximum inhibitory effect of dexamethasone on IL-1α-induced IL-6 and CXCL8 production. The impaired glucocorticoid response was detected with 4 h of TGF-ß (40 pM) exposure (and 4 h IL-1α to induce CXCL8 expression) and therefore was not secondary to EMT, a process that requires longer incubation periods and higher concentrations of TGF-ß. TGF-ß also impaired dexamethasone regulation of granulocyte-macrophage colony-stimulating factor in thrombin-stimulated BEAS-2B epithelial cells. Impaired regulation of CXCL8 was associated with markedly reduced GRE transactivation and reduced induction of mRNA for IκBα, the glucocorticoid-inducible leucine zipper and the epithelial sodium channel (SCNN1A). The expression, cellular levels and nuclear localization of GRα were reduced by TGF-ß. CONCLUSIONS AND IMPLICATIONS: We have identified mechanisms underlying the impairment of responses to glucocorticoids by TGF-ß in the A549 and BEAS-2B cell lines.

In vitro and in vivo evidence for anti-inflammatory properties of 2-methoxyestradiol.

2-Methoxyestradiol (2MEO) is an endogenous metabolite of 17ß-estradiol that interacts with estrogen receptors and microtubules. It has acute anti-inflammatory activity in animal models that is not attributable to known antiproliferative or antiangiogenic actions. Because macrophages are central to the innate inflammatory response, we examined whether suppression of macrophage activation by 2MEO could account for some of its anti-inflammatory effects. Inflammatory mediator production stimulated by lipopolysaccharide (LPS) and interferon-γ in the J774 murine macrophage cell line or human monocytes was measured after treatment with 2MEO or the anti-inflammatory agent dexamethasone. The effect of these agents on LPS-induced acute lung inflammation in mice was also examined. 2MEO suppressed J774 macrophage interleukin-6 and prostaglandin E2 production (by 30 and 47%, respectively, at 10 µM) and human monocyte tumor necrosis factor-α production (by 60% at 3 µM). Estradiol had no effect on J774 macrophage activation, nor did the estrogen receptor antagonist 7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17ß-diol (ICI 182,780) prevent the effects of 2MEO. The actions of 2MEO were not mimicked by the microtubule-interfering agents colchicine or paclitaxel. In mice exposed to LPS, bronchoalveolar lavage protein content, a measure of vascular leak and epithelial injury, was reduced to a comparable extent (~54%) by treatment with 2MEO (150 mg · kg⁻¹) or dexamethasone (1 mg · kg⁻¹). In addition, 2MEO reduced LPS-induced interleukin-6 gene expression. Thus, 2MEO modulates macrophage activation in vitro and has high-dose acute anti-inflammatory activity in vivo. These findings are consistent with the acute anti-inflammatory actions of 2MEO being mediated in part by the suppression of macrophage activation.

Collagen impairs glucocorticoid actions in airway smooth muscle through integrin signalling.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: ASM cultured from human airways has been seeded on to either type I monomeric collagen or a laminin pentapeptide, YIGSR. The role of alpha2beta1 integrin in the collagen-induced glucocorticoid resistance was investigated using a function blocking monoclonal antibody. KEY RESULTS: Culture of ASM on collagen I, but not laminin, led to a greater proliferative response that was insensitive to regulation by dexamethasone (100 nM). The anti-migratory effects of the glucocorticoid, fluticasone propionate (1 nM) were also impaired by contact of ASM with collagen. The impaired anti-mitogenic action of dexamethasone was associated with a failure to reduce the levels of the rate-limiting cell cycle regulatory protein, cyclin D1. When signalling through the alpha2beta1 integrin was reduced, dexamethasone-mediated reductions in proliferation and cyclin D1 levels were restored. CONCLUSIONS AND IMPLICATIONS: In the collagen-rich microenvironment of the inflamed and fibrotic asthmatic airway, integrin/ECM interactions may contribute to glucocorticoid resistance.

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.