Ferrous citrate up-regulates the NOS2 through nuclear translocation of NFκB induced by free radicals generation in mouse cerebral endothelial cells

PLoS One. 2012;7(9):e46239. doi: 10.1371/journal.pone.0046239. Epub 2012 Sep 28.


Previous studies indicate that the inducible nitric oxide synthase 2 (NOS2) of the brain vascular tissue in experimental subarachnoid hemorrhage (SAH) rats is a critical factor for inducing cerebral vasospasm. However, the underlying molecular mechanisms remain to be elucidated. Here, we applied ferrous citrate (FC) complexes to the primary cultured mouse cerebral endothelial cell (CEC) to mimic the SAH conditions and to address the issue how SAH-induced NOS2 up-regulation. Using immunocytochemical staining technique, we demonstrated that NOS2 was expressed in the cultured CEC. Treatment of the CEC with FC induced increases of the intracellular level of ROS, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) nuclear translocation as well as NFκB binding onto the NOS promoter, and the levels of NOS2 mRNA and protein. These effects were abolished by pre-treatment of the cell with N-Acetyl-Cysteine (NAC), a reactive oxygen species (ROS) scavenger. In the present study, two previously predicted NFκB binding sites were confirmed in the NOS2 promoter within the range of -1529 bp to -1516 bp and -1224 bp to -1210 bp. Interestingly, both NFκB binding sites are involved in the FC-activated NOS2 transcriptional activity; the binding site located at -1529 bp to -1516 bp played a greater role than the other binding site located at -1224 bp to -1210 bp in the mouse CEC. These findings highlight the molecular mechanism underlying FC-induced up-regulation of NOS2 in the mouse CEC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / pharmacology
  • Animals
  • Binding Sites
  • Cell Nucleus / metabolism
  • Cells, Cultured
  • Cerebral Cortex / cytology
  • Cerebral Cortex / drug effects
  • Cerebral Cortex / metabolism
  • Endothelial Cells / cytology
  • Endothelial Cells / drug effects*
  • Endothelial Cells / metabolism
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism
  • Ferrous Compounds / pharmacology*
  • Free Radical Scavengers / pharmacology
  • Free Radicals / agonists
  • Free Radicals / antagonists & inhibitors
  • Free Radicals / metabolism*
  • Male
  • Mice
  • NF-kappa B / genetics
  • NF-kappa B / metabolism*
  • Nitric Oxide Synthase Type II / genetics
  • Nitric Oxide Synthase Type II / metabolism*
  • Promoter Regions, Genetic*
  • Protein Binding
  • Protein Transport
  • RNA, Messenger / biosynthesis
  • Transcription, Genetic / drug effects
  • Up-Regulation / drug effects


  • Ferrous Compounds
  • Free Radical Scavengers
  • Free Radicals
  • NF-kappa B
  • RNA, Messenger
  • monoferrous acid citrate
  • Nitric Oxide Synthase Type II
  • Acetylcysteine

Grant support

This work was supported by research grants from the National Science Council of Taiwan (NSC-101-2320-B-038-005 and NSC-101-2629-B-037-001 to W.S.L.; NSC-95-2320-B-037-032-MY2 and NSC97-2314-B-037-039-MY2 to C.H.; NSC-95-2320-B-038-027-MY2 to C.C.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.