Agonist binding to beta-adrenergic receptors on human airway epithelial cells inhibits migration and wound repair

摘要

Human airway epithelial cells express beta-adrenergic receptors (beta-ARs), which regulate mucociliary clearance by stimulating transepithelial anion transport and ciliary beat frequency. Previous studies using airway epithelial cells showed that stimulation with isoproterenol increased cell migration and wound repair by a cAMP-dependent mechanism. In the present study, impedance-sensing arrays were used to measure cell migration and epithelial restitution following wounding of confluent normal human bronchial epithelial (NHBE) and Calu-3 cells by electroporation. Stimulation with epinephrine or the beta_2-AR-selective agonist salbuta-mol significantly delayed wound closure and reduced the mean surface area of lamellipodia protruding into the wound. Treatment with the beta-AR bias agonist carvedilol or isoetharine also produced a delay in epithelial restitution similar in magnitude to epinephrine and salbutamol. Measurements of extracellular signal-regulated kinase phosphorylation following salbutamol or carvedilol stimulation showed no significant change in the level of phosphorylation compared with untreated control cells. However, inhibition of protein phosphatase 2A activity completely blocked the delay in wound closure produced by beta-AR agonists. In Calu-3 cells, where CFTR expression was inhibited by RNAi, salbutamol did not inhibit wound repair, suggesting that beta-AR agonist stimulation and loss of CFTR function share a common pathway leading to inhibition of epithelial repair. Confocal images of the basal membrane of Calu-3 cells labeled with anti-beta_1-integrin (clone HUTS-4) antibody showed that treatment with epinephrine or carvedilol reduced the level of activated integrin in the membrane. These findings suggest that treatment with 3-AR agonists delays airway epithelial repair by a G protein- and cAMP-independent mechanism involving protein phosphatase 2A and a reduction in beta_1-integrin activation in the basal membrane.