Transcriptional suppression of CYP2A13 expression by lipopolysaccharide in cultured human lung cells and the lungs of a CYP2A13-humanized mouse model

Toxicol Sci. 2013 Oct;135(2):476-85. doi: 10.1093/toxsci/kft165. Epub 2013 Jul 24.

Abstract

CYP2A13, a human P450 enzyme preferentially expressed in the respiratory tract, is highly efficient in the metabolic activation of tobacco-specific nitrosamines. The aim of this study was to test the hypothesis that inflammation suppresses CYP2A13 expression in the lung, thus explaining the large interindividual differences in CYP2A13 levels previously found in human lung biopsy samples. We first demonstrated that the bacterial endotoxin lipopolysaccharide (LPS) and the proinflammatory cytokine IL-6 can suppress CYP2A13 messenger RNA (mRNA) expression in the NCI-H441 human lung cell line. We then report that an ip injection of LPS (1mg/kg), which induces systemic and lung inflammation, caused substantial reductions in CYP2A13 mRNA (~50%) and protein levels (~80%) in the lungs of a newly generated CYP2A13-humanized mouse model. We further identified two critical CYP2A13 promoter regions, one (major) between -484 and -1008bp and the other (minor) between -134 and -216bp, for the response to LPS, through reporter gene assays in H441 cells. The potential involvement of the nuclear factor NF-κB in LPS-induced CYP2A13 downregulation was suggested by identification of putative NF-κB binding sites within the LPS response regions and effects of an NF-κB inhibitor (pyrrolidine dithiocarbamate) on CYP2A13 expression in H441 cells. Results from gel shift assays further confirmed binding of NF-κB-like nuclear proteins of H441 cells to the major LPS response region of the CYP2A13 promoter. Thus, our findings strongly support the hypothesis that CYP2A13 levels in human lung can be suppressed by inflammation associated with disease status in tissue donors, causing underestimation of CYP2A13 levels in healthy lung.

Keywords: CYP2A; LPS; chemical carcinogenesis.; inflammation; lung.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Aryl Hydrocarbon Hydroxylases / genetics*
  • Cells, Cultured
  • Humans
  • Lipopolysaccharides / toxicity*
  • Mice
  • Promoter Regions, Genetic
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Transcription, Genetic*

Substances

  • Lipopolysaccharides
  • RNA, Messenger
  • Aryl Hydrocarbon Hydroxylases
  • CYP2A13 protein, human