title: Modulation of inducible nitric oxide synthase expression by sumoylation.
abstract: BACKGROUND
  In astrocytes, the inflammatory induction of Nitric Oxide Synthase type 2 (NOS2) is inhibited by noradrenaline (NA) at the transcriptional level however its effects on specific transcription factors are not fully known. Recent studies show that the activity of several transcription factors including C/EBPbeta, which is needed for maximal NOS2 expression, is modulated by conjugation of the small molecular weight protein SUMO. We examined whether the expression of SUMO Related Genes (SRGs: SUMO-1, the conjugating enzyme Ubc9, and the protease SENP1) are affected by inflammatory conditions or NA and whether SUMO-1 regulates NOS2 through interaction with C/EBPbeta.
  
  
  METHODS
  Bacterial endotoxin lipopolysaccharide (LPS) was used to induce inflammatory responses including NOS2 expression in primary astrocytes. The mRNA levels of SRGs were determined by QPCR. A functional role for SUMOylation was evaluated by determining effects of over-expressing SRGs on NOS2 promoter and NFkappaB binding-element reporter constructs. Interactions of SUMO-1 and C/EBPbeta with the NOS2 promoter were examined by chromatin immunoprecipitation assays. Interactions of SUMO-1 with C/EBPbeta were examined by immunoprecipitation and Western blot analysis and by fluorescence resonance energy transfer (FRET) assays.
  
  
  RESULTS
  LPS decreased mRNA levels of SUMO-1, Ubc9 and SENP1 in primary astrocytes and a similar decrease occurred during normal aging in brain. NA attenuated the LPS-induced reductions and increased SUMO-1 above basal levels. Over-expression of SUMO-1, Ubc9, or SENP1 reduced the activation of a NOS2 promoter, whereas activation of a 4 x NFkappaB binding-element reporter was only reduced by SUMO-1. ChIP studies revealed interactions of SUMO-1 and C/EBPbeta with C/EBP binding sites on the NOS2 promoter that were modulated by LPS and NA. SUMO-1 co-precipitated with C/EBPbeta and a close proximity was confirmed by FRET analysis.
  
  
  CONCLUSIONS
  Our results demonstrate that SUMOylation regulates NOS2 expression in astrocytes, and point to modification of C/EBPbeta as a possible mechanism of action. Targeting the SUMOylation pathway may therefore offer a novel means to regulate inflammatory NOS2 expression in neurological conditions and diseases.
authors.affiliation: Department of Anesthesiology, University of Illinois, Chicago, IL 60612, USA. candan@uic.edu
authors.forename: Candan A
authors.initials: CA
authors.lastname: Akar
authors.forename: Douglas L
authors.initials: DL
authors.lastname: Feinstein