title: A critical role for increased labile zinc in reducing sensitivity of cultured sheep pulmonary artery endothelial cells to LPS-induced apoptosis.
abstract: We previously noted an important signaling role for decreased labile intracellular zinc ([ Zn ] (i)) in LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) (Tang ZL, Wasserloos KJ, Liu X, Stitt MS, Reynolds IJ, Pitt BR, St Croix CM. Mol Cell Biochem 234-235: 211-217, 2002; Thambiayya K, Wasserloos KJ, Huang Z, Kagan VE, St Croix CM, Pitt BR. Am J Physiol Lung Cell Mol Physiol 300: L624-632, 2011). In the present study, we used small interfering RNA (siRNA) to important contributors of zinc homeostasis [ SLC39A14 or Zrt/Irt-like protein 14 (ZIP14), a zinc importer; metallothionein (MT), a zinc binding protein ] to define molecular pathways by which extracellular zinc or nitric oxide (NO) increase labile [ Zn ] (i) [ e.g., zinc-sensitive fluorophore (FluoZin-3) detectable and/or chelatable by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine ] and reduce the sensitivity of SPAEC to LPS. Addition of 10 μM zinc to serum-free medium of SPAEC increased [ Zn ] (i) and abolished LPS-induced apoptosis (e.g., increased annexin V binding). The increase in [ Zn ] (i) and the protective effect of extracellular zinc were sensitive to reduction in ZIP14 expression (by siRNA), but not affected by collectively knocking down major isoforms of sheep MT (sMT-Ia, -Ib, -Ic, and -II). Pretreatment of wild-type SPAEC with 250 μM of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased labile zinc in a relatively similar fashion to addition of extracellular zinc and reduced sensitivity of SPAEC to LPS-induced apoptosis (e.g., caspase-3/7 activation) in a N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine-sensitive fashion. The antiapoptotic effects of SNAP were insensitive to siRNA knockdown of ZIP14, but were abolished (along with SNAP-induced increase in [ Zn ] (i)) when SPAEC were pretreated with siRNA to sheep MT. Zinc was able to directly inhibit recombinant caspase-3 activity in an in vitro assay. Collectively, these data show that increases in labile [ Zn ] (i) are an important component of ZIP14- or NO-mediated resistance to LPS-induced apoptosis. Cytoprotection via ZIP14 appeared to be secondary to transcellular movement of extracellular zinc, whereas NO-mediated protection was secondary to S-nitrosation of MT and redistribution of [ Zn ] (i).
authors.affiliation: Dept. Environmental and Occupational Health, Univ. of Pittsburgh Graduate School Public Health, Bridgeside Point Bldg., 100 Technology Dr., Ste. 555, Pittsburgh, PA 15219, USA.
authors.forename: Kalidasan
authors.initials: K
authors.lastname: Thambiayya
authors.forename: Karla
authors.initials: K
authors.lastname: Wasserloos
authors.forename: Valerian E
authors.initials: VE
authors.lastname: Kagan
authors.forename: Detcho
authors.initials: D
authors.lastname: Stoyanovsky
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Pitt
mesh_terms: D000818:Animals; D017209:Apoptosis; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D008070:Lipopolysaccharides; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D012756:Sheep; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; C017800:zinc-binding protein; D008668:Metallothionein; D053148:Caspase 3; D015032:Zinc