[PMC free article] [PubMed] [CrossRef] [Google Scholar] 19. inflammatory responses similar to those induced by IL-13. Furin inactivation or IL-13 caused talin cleavage and integrin inactivation, resulting in the inactivation of vinculin and paxillin. Paxillin inactivation resulted in the coupling of Akt to -parvin IPP complexes, which catalyze Akt activation and an inflammatory response. The results demonstrate that furin inhibits inflammation in airway smooth muscle induced by IL-13 and that the anti-inflammatory effects of furin are mediated by activating integrin proteins and integrin-associated signaling complexes that regulate Akt-mediated pathways to the nucleus. Furin may have therapeutic potential for the treatment of inflammatory conditions of the lungs and airways. = 0.0001) (= 0.0001) (the depletion of furin caused a significant increase in eotaxin secretion (= 0.0272)= 0.0001). Furin inhibition also significantly potentiated IL-13-induced Akt phosphorylation (= 0.019). Data were analyzed by using a paired Students test (= 0.0001). rFurin also increased integrin activation in the presence of IL-13 (= 0.0001). = 0.0004; paxillin: = 0.0001). rFurin also increased vinculin and paxillin phosphorylation in the PHF9 presence of IL-13 (vinculin: = 0.0025; paxillin: = 0.0002). Data were analyzed by using one-way ANOVA. All values are means SE. *Significant difference between groups. rF, rFurin. Open in a separate window Figure 5. Furin Inhibitor II inhibits the activation of 1-integrins and integrin-associated adhesion proteins by acetylcholine (Ach) in airway smooth muscle tissues. = 0.0001) and incubation with Furin Inhibitor II inhibited the increase of activated 1-integrin induced by ACh (= 0.0001). = 0.0001; paxillin: = 0.0001). Data were analyzed by using one-way ANOVA. All values are means SE. *Significant difference between groups. Inh, Furin Inhibitor II. Other materials and reagents used were as follows: recombinant canine IL-13, recombinant human furin (rhFurin), and human eotaxin-1 ELISA kit (R&D Systems) and Furin inhibitor II (Sigma). Furin shRNA plasmids (Cat. No. SC-40595) were purchased from Santa Cruz Biotechnology, (Dallas, TX). Preparation of Smooth Muscle Tissues All procedures were in accordance with the procedures approved by the Institutional Animal Care and Use Committee (IUCAC) of Desformylflustrabromine HCl Indiana University School of Medicine under the National Research Councils Guide for the Care and Use of Laboratory Animals. The Indiana University Laboratory Animal Resource Center (LARC) at Indiana University School of Medicine procured mongrel dogs (20C30?kg, either sex) from LBL Kennels, Reelsville, Indiana. LARC personnel euthanized animals by an intravenous injection of Fatal-Plus (Vortech Pharmaceuticals, Ltd, Dearborn, MI) [pentobarbital sodium, 390 mg/mL; propylene glycol, 0.01 mg/mL; ethyl alcohol, 0.29 mg/mL; benzyl alcohol (preservative), 0.2 mg/mL] at a dose of 0.3 mL/kg. A tracheal segment was immediately removed and immersed in physiological saline solution (composition in mM: Desformylflustrabromine HCl 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose). Strips of tracheal smooth muscle (in mm: 1.0 wide 0.2C0.5 thick 15 long) Desformylflustrabromine HCl were dissected free of connective and epithelial tissues and maintained within a tissue bath at 37C. Muscle tissues were equilibrated and attached to metal mounts to maintain them at a constant length. Tissues were treated overnight with 100?M Furin Inhibitor II or 0.5?g/mL recombinant furin (rFurin). Furin Inhibitor II (Hexa-d-arginine) is a polyarginine peptide that acts as a competitive inhibitor of furin (40C42). Tissues were treated with furin shRNA plasmids to deplete endogenous furin. Tissues were stimulated for 30?min or incubated overnight with 50 ng/mL IL-13 to elicit inflammatory responses (1, 7). Tissues were treated for 5?min with 10?5 M acetylcholine (ACh) to elicit contractile responses. Immunoblots Muscle tissues were pulverized in liquid N2 using a mortar and pestle before the extraction of proteins. Proteins were extracted from pulverized muscle tissues for electrophoresis and Western blotting using a buffer containing 20?mM Tris-HCl at pH.
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