Terry Machen
Lab Personnel

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  Zhu Fu, K Bettega, S Carroll, KR Buchholz, Terry Machen (2006). Role of Ca in responses of airway epithelia to P. aeruginosa, flagellin, ATP and thapsigargin. Amer. J. Physiol.,Lung Cell Mol Physiol. 292, L353-64.

     Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca2+ concentration ([Ca]i) in airway epithelial cell lines JME and Calu-3, but bacteria or flagellin activated NF-{kappa}B, IL-8 promoter, and IL-8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca2+ pump, releases endoplasmic reticulum Ca2+, and triggers Ca2+ entry through plasma membrane channels) both increased [Ca]i but hardly stimulated NF-{kappa}B and IL-8. ATP and thapsigargin elicited larger, synergistic activations of NF-{kappa}B and IL-8 secretion when combined with flagellin. BAPTA-AM (to buffer [Ca]i) or Ca2+-free solution reduced increases in [Ca]i due to ATP or thapsigargin and also reduced NF-{kappa}B activation and IL-8 secretion triggered by flagellin, ATP, thapsigargin, ATP + flagellin, and thapsigargin + flagellin. IL-8 promoter analysis showed that AP-1 and CCAAT/enhancer-binding protein (C/EBP)beta/nuclear factor for IL-6 (NF-IL6) sites were important for IL-8 expression, and the NF-{kappa}B-binding site was critical for activation by all agonists and for activation by [Ca]i. Thus increased [Ca]i was not required for P. aeruginosa- or flagellin-activated NF-{kappa}B and IL-8 expression and secretion, and increased [Ca]i was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in [Ca]i synergized with flagellin or P. aeruginosa, and buffering or reducing [Ca]i reduced these responses. Thus [Ca]i plays an important regulatory role in P. aeruginosa- or flagellin-activated innate immune responses in airway epithelia. Dose-dependent responses indicated that flagellin-ATP synergism occurred most prominently at ATP concentrations ([ATP]) > 10 M and [flagellin] >108 g/ml and during steady increases rather than oscillations in [Ca]i.