Modulation of inducible nitric oxide synthase expression by sumoylation

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.

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.

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.

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.