Role of Ca2+ in responses of airway epithelia to Pseudomonas aeruginosa, flagellin, ATP, and thapsigargin.
Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca(2+) concentration ([Ca](i)) in airway epithelial cell lines JME and Calu-3, but bacteria or flagellin activated NF-kappaB, IL-8 promoter, and IL-8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca(2+) pump, releases endoplasmic reticulum Ca(2+), and triggers Ca(2+) entry through plasma membrane channels) both increased [Ca](i) but hardly stimulated NF-kappaB and IL-8. ATP and thapsigargin elicited larger, synergistic activations of NF-kappaB and IL-8 secretion when combined with flagellin. BAPTA-AM (to buffer [Ca](i)) or Ca(2+)-free solution reduced increases in [Ca](i) due to ATP or thapsigargin and also reduced NF-kappaB 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-kappaB-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-kappaB 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 microM and [flagellin] >10(-8) g/ml and during steady increases rather than oscillations in [Ca](i).
Fu
Zhu
Z
Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA 94720-3200, USA.
Bettega
Kelly
K
Carroll
Susheela
S
Buchholz
Kerry R
KR
Machen
Terry E
TE
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001483:Base Sequence; D020013:Calcium Signaling; D002460:Cell Line; D003550:Cystic Fibrosis / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D005408:Flagellin / Q000494:pharmacology; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000472:pathogenicity*; D012137:Respiratory System / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology*; D019284:Thapsigargin / Q000494:pharmacology
D017931:DNA Primers; D016209:Interleukin-8; D016328:NF-kappa B; D005408:Flagellin; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019284:Thapsigargin; D000255:Adenosine Triphosphate