LPS-induced decrease in intracellular labile zinc, [Zn]i, contributes to apoptosis in cultured sheep pulmonary artery endothelial cells.
A role in signal transduction for a vanishingly small labile pool of intracellular zinc ([Zn](i)) has been inferred by the sensitivity of various physiological pathways to zinc chelators such as N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and/or associations with changes in nonprotein-bound zinc-sensitive fluorophores. Although we (44) reported that LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) was exacerbated by TPEN, 1) we did not detect acute (30 min) changes in [Zn](i), and 2) it is unclear from other reports whether LPS increases or decreases [Zn](i) and whether elevations or decreases in [Zn](i) are associated with cell death and/or apoptosis. In the present study, we used both chemical (FluoZin-3 via live cell epifluorescence microscopy and fluorescence-activated cell sorting) and genetic (luciferase activity of a chimeric reporter encoding zinc-sensitive metal-response element and changes in steady-state mRNA of zinc importer, SLC39A14 or ZIP14) techniques to show that LPS caused a delayed time-dependent (2-4 h) decrease in [Zn](i) in SPAEC. A contributory role of decreases in [Zn](i) in LPS-induced apoptosis (as determined by caspase-3/7 activation, annexin-V binding, and cytochrome c release) in SPAECs was revealed by mimicking the effect of LPS with the zinc chelator, TPEN, and inhibiting LPS- (or TPEN)-induced apoptosis with exogenous zinc. Collectively, these are the first data demonstrating a signaling role for decrease in [Zn](i) in pulmonary endothelial cells and suggest that endogenous levels of labile zinc may affect sensitivity of pulmonary endothelium to the important and complex proapoptotic stimulus of LPS.
Department of Bioengineering, University of Pittsburgh and Universityof Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, USA.
Kalidasan
K
Thambiayya
Karla J
KJ
Wasserloos
Zhentai
Z
Huang
Valerian E
VE
Kagan
Claudette M
CM
St Croix
Bruce R
BR
Pitt
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D017930:Genes, Reporter; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D015854:Up-Regulation / Q000187:drug effects; D015032:Zinc / Q000378:metabolism*
D027682:Cation Transport Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D053148:Caspase 3; D053179:Caspase 7; D015032:Zinc
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