LPS-induced decrease in intracellular labile zinc, [Zn]i, contributes to apoptosis in cultured sheep pulmonary artery endothelial cells

Kalidasan Thambiayya, Karla J. Wasserloos, Zhentai Huang, Valerian E. Kagan, Claudette M. St. Croix, Bruce R. Pitt

Abstract

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.

  • pulmonary endothelium
  • lipopolysaccharide
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