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Functional analysis of the antioxidant paraoxonase-3 in lung epithelial cells by rna interference
  1. G Belteki1,2,
  2. S Kempster2,
  3. D Licence2,
  4. D S Charnock-Jones2,
  5. G C S Smith2
  1. 1Neonatal Intensive Care Unit, The Rosie Hospital, Cambridge, UK
  2. 2Department of Obstetrics and Gynaecology, University of Cambridge, Cambridge, UK

Abstract

Background The lung undergoes physiologic changes during late gestation in order to prepare for its postnatal environment and function. This comprises up-regulation of antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase. Recently we have described perinatal up-regulation of paraoxonase-3 (PON3), an uncharacterized antioxidant gene, in rodent and ovine lungs. In the current study we analysed the functional significance of PON3 expression.

Methods We applied RNA interference (RNAi) to reduce gene expression in A549 cells, a human lung epithelial cell line resembling type 2 pneumocytes. Transient and permanent RNAi was achieved by transfection of short interfering or short hairpin RNAs, respectively. To mimic fetal environment cells were cultured in 2% oxygen. Gene expression was analysed by quantitative PCR. IL-8 secretion was analysed by ELISA. Total antioxidant activity was determined in Trolox units.

Results siRNAs reduced PON3 expression by 77% (p<0.05) and 69% (p<0.001) in fetal or postnatal (ambient) oxygen concentration, respectively. There was a significant difference in antioxidant capacity between knock-down and control cells. PON3 silencing increased the expression of the gene for heat-shock protein HSP5A by 25% in postnatal oxygen levels; this up-regulation was absent when cells were cultured in low oxygen. Transient or stable silencing did not affect cell viability or the response to H2O2-mediated oxidative stress; however, stable silencing resulted in slower cell proliferation.

Conclusion Absence of PON3 causes reduced proliferation and endoplasmic reticulum stress in lung cells. PON3 may be required to protect the lung from consequences of postnatal rise in oxygen concentration.

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