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Am J Physiol Lung Cell Mol Physiol (September 8, 2006). doi:10.1152/ajplung.00042.2006
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Submitted on February 1, 2006
Accepted on August 31, 2006

Role of Ca2+ in Responses of Airway Epithelia to P. aeruginosai, flagellin, ATP and Thapsigargin

Zhu Fu1, Kelly Bettega1, Susheela Carroll1, Kerry R Buchholz2, and Terry E Machen1*

1 Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, United States
2 Program in Infectious Diseases and Immunology, University of California at Davis, Berkeley, California, United States

* To whom correspondence should be addressed. E-mail: tmachen{at}berkeley.edu.

Neither P. aeruginosa nor flagellin affected cytosolic [Ca2+], Cai, in airway epithelial cell lines JME or Calu-3, but bacteria or flagellin activated NF-{kappa}B, IL8 promoter and IL8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca2+ pump, releases endoplasmic reticulum Ca2+ and triggers Ca2+ entry through plasma membrane channels) both increased Cai but hardly stimulated NF-{kappa}B and IL8. ATP and thapsigargin elicited larger, synergistic activations of NF-{kappa}B and IL8 secretion when combined with flagellin. BAPTA/AM (to buffer Cai) or Ca2+-free solution reduced increases in Cai due to ATP or thapsigargin and also reduced NF-{kappa}B activation and IL8 secretion triggered by flagellin, ATP, thapsigargin, ATP+flagellin and thapsigargin+flagellin. IL8 promoter analysis showed that AP-1 and CEBP{beta}/NF-IL6 sites were important for IL8 expression, and the NF-{kappa}B-binding site was critical for activation by all agonists and for activation by Cai. Thus, increased Cai was not required for P. aeruginosa- or flagellin-activated NF-{kappa}B and IL8 expression and secretion, and increased Cai was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in Cai synergized with flagellin or P. aeruginosa, and buffering or reducing Cai reduced these responses. Thus, Cai 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] > 10 µM and [flagellin] >10-8 g/ml and during steady increases rather than oscillations in Cai.




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