Pharmacology and Therapeutics - Research Publications

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    Plasminogen-stimulated airway smooth muscle cell proliferation is mediated by urokinase and annexin A2, involving plasmin-activated cell signalling
    Stewart, AG ; Xia, YC ; Harris, T ; Royce, S ; Hamilton, JA ; Schuliga, M (WILEY-BLACKWELL, 2013-12)
    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.
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    Plasminogen-Stimulated Inflammatory Cytokine Production by Airway Smooth Muscle Cells Is Regulated by Annexin A2
    Schuliga, M ; Langenbach, S ; Xia, YC ; Qin, C ; Mok, JSL ; Harris, T ; Mackay, GA ; Medcalf, RL ; Stewart, AG (AMER THORACIC SOC, 2013-11)
    Plasminogen has a role in airway inflammation. Airway smooth muscle (ASM) cells cleave plasminogen into plasmin, a protease with proinflammatory activity. In this study, the effect of plasminogen on cytokine production by human ASM cells was investigated in vitro. Levels of IL-6 and IL-8 in the medium of ASM cells were increased by incubation with plasminogen (5-50 μg/ml) for 24 hours (P < 0.05; n = 6-9), corresponding to changes in the levels of cytokine mRNA at 4 hours. The effects of plasminogen were attenuated by α2-antiplasmin (1 μg/ml), a plasmin inhibitor (P < 0.05; n = 6-12). Exogenous plasmin (5-15 mU/ml) also stimulated cytokine production (P < 0.05; n = 6-8) in a manner sensitive to serine-protease inhibition by aprotinin (10 KIU/ml). Plasminogen-stimulated cytokine production was increased in cells pretreated with basic fibroblast growth factor (300 pM) in a manner associated with increases in urokinase plasminogen activator expression and plasmin formation. The knockdown of annexin A2, a component of the putative plasminogen receptor comprised of annexin A2 and S100A10, attenuated plasminogen conversion into plasmin and plasmin-stimulated cytokine production by ASM cells. Moreover, a role for annexin A2 in airway inflammation was demonstrated in annexin A2-/- mice in which antigen-induced increases in inflammatory cell number and IL-6 levels in the bronchoalveolar lavage fluid were reduced (P < 0.01; n = 10-14). In conclusion, plasminogen stimulates ASM cytokine production in a manner regulated by annexin A2. Our study shows for the first time that targeting annexin A2-mediated signaling may provide a novel therapeutic approach to the treatment of airway inflammation in diseases such as chronic asthma.
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    Transforming Growth Factor-β-Induced Differentiation of Airway Smooth Muscle Cells Is Inhibited by Fibroblast Growth Factor-2
    Schuliga, M ; Javeed, A ; Harris, T ; Xia, Y ; Qin, C ; Wang, Z ; Zhang, X ; Lee, PVS ; Camoretti-Mercado, B ; Stewart, AG (AMER THORACIC SOC, 2013-03)
    In asthma, basic fibroblast growth factor (FGF-2) plays an important (patho)physiological role. This study examines the effects of FGF-2 on the transforming growth factor-β (TGF-β)-stimulated differentiation of airway smooth muscle (ASM) cells in vitro. The differentiation of human ASM cells after incubation with TGF-β (100 pM) and/or FGF-2 (300 pM) for 48 hours was assessed by increases in contractile protein expression, actin-cytoskeleton reorganization, enhancements in cell stiffness, and collagen remodeling. FGF-2 inhibited TGF-β-stimulated increases in transgelin (SM22) and calponin gene expression (n = 15, P < 0.01) in an extracellular signal-regulated kinase 1/2 (ERK1/2) signal transduction-dependent manner. The abundance of ordered α-smooth muscle actin (α-SMA) filaments formed in the presence of TGF-β were also reduced by FGF-2, as was the ratio of F-actin to G-actin (n = 8, P < 0.01). Furthermore, FGF-2 attenuated TGF-β-stimulated increases in ASM cell stiffness and the ASM-mediated contraction of lattices, composed of collagen fibrils (n = 5, P < 0.01). However, the TGF-β-stimulated production of IL-6 was not influenced by FGF-2 (n = 4, P > 0.05), suggesting that FGF-2 antagonism is selective for the regulation of ASM cell contractile protein expression, organization, and function. Another mitogen, thrombin (0.3 U ml(-1)), exerted no effect on TGF-β-regulated contractile protein expression (n = 8, P > 0.05), α-SMA organization, or the ratio of F-actin to G-actin (n = 4, P > 0.05), suggesting that the inhibitory effect of FGF-2 is dissociated from its mitogenic actions. The addition of FGF-2, 24 hours after TGF-β treatment, still reduced contractile protein expression, even when the TGF-β-receptor kinase inhibitor, SB431542 (10 μM), was added 1 hour before FGF-2. We conclude that the ASM cell differentiation promoted by TGF-β is antagonized by FGF-2. A better understanding of the mechanism of action for FGF-2 is necessary to develop a strategy for therapeutic exploitation in the treatment of asthma.