title: Thapsigargin blocks Pseudomonas aeruginosa homoserine lactone-induced apoptosis in airway epithelia.
abstract: Pseudomonas aeruginosa secretes N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression. Micromolar concentrations are found in the airway surface liquid of infected lungs. Exposure of the airway surface to C12 caused a loss of transepithelial resistance within 1 h that was accompanied by disassembly of tight junctions, as indicated by relocation of the tight junction protein zonula occludens 1 from the apical to the basolateral pole and into the cytosol of polarized human airway epithelial cell cultures (Calu-3 and primary tracheal epithelial cells). These effects were blocked by carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone, a pan-caspase blocker, indicating that tight junction disassembly was an early event in C12-triggered apoptosis. Short-duration (10 min) pretreatment of airway epithelial (Calu-3 and JME) cells with 1 μM thapsigargin (Tg), an inhibitor of Ca(2+) uptake into the endoplasmic reticulum (ER), was found to be protective against the C12-induced airway epithelial barrier breakdown and also against other apoptosis-related effects, including shrinkage and fragmentation of nuclei, activation of caspase 3/7 (the executioner caspase in apoptosis), release of ER-targeted redox-sensitive green fluorescent protein into the cytosol, and depolarization of mitochondrial membrane potential. Pretreatment of Calu-3 airway cell monolayers with BAPTA-AM [to buffer cytosolic Ca(2+) concentration (Cacyto)] or Ca(2+)-free solution + BAPTA-AM reduced C12 activation of apoptotic events, suggesting that C12-triggered apoptosis may involve Ca(2+). Because C12 and Tg reduced Ca(2+) concentration in the ER and increased Cacyto, while Tg increased mitochondrial Ca(2+) concentration (Camito) and C12 reduced Camito, it is proposed that Tg may reduce C12-induced apoptosis in host cells not by raising Cacyto, but by preventing C12-induced decreases in Camito.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California; and.
authors.forename: Christian
authors.initials: C
authors.lastname: Schwarzer
authors.forename: Bharat
authors.initials: B
authors.lastname: Ravishankar
authors.forename: Maria
authors.initials: M
authors.lastname: Patanwala
authors.forename: Stacey
authors.initials: S
authors.lastname: Shuai
authors.forename: Zhu
authors.initials: Z
authors.lastname: Fu
authors.forename: Beate
authors.initials: B
authors.lastname: Illek
authors.forename: Horst
authors.initials: H
authors.lastname: Fischer
authors.forename: Terry E
authors.initials: TE
authors.lastname: Machen
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D019610:Cytoprotection; D017097:Electric Impedance; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation; D006801:Humans; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D061251:Primary Cell Culture; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology*; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D014132:Trachea / Q000187:drug effects* / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: D002614:Chelating Agents; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C088386:homoserine lactone; D019284:Thapsigargin; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D015107:4-Butyrolactone; D002118:Calcium
keywords: calcium; caspase; endoplasmic reticulum; innate immunity; mitochondria; tight junctions; zonula occludens 1