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Modulation of inducible nitric oxide synthase expression by sumoylation.

Akar CA, Feinstein DL - J Neuroinflammation (2009)

Bottom Line: 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.Our results demonstrate that SUMOylation regulates NOS2 expression in astrocytes, and point to modification of C/EBPbeta as a possible mechanism of action.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, University of Illinois, Chicago, IL 60612, USA. candan@uic.edu

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.

Conclusion: 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.

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Sumoylation of C/EBPβ in astrocytes. Primary rat astrocytes were co-transfected with YFP-SUMO-1 and CFP-C/EBPβ expression plasmids. After 24 hr the cells were fixed, and cells co-expressing YFP-SUMO-1 and CFP-C/EBPβ were subjected to FRET analysis. (A) Representative image of CFP and YFP fluorescence after linear un-mixing of emission spectra and merged image showing the region of interest (ROI) analyzed. (B) FRET data after acceptor photobleaching of the ROI indicated ("1") in panel A, showing an increase in CFP fluorescence coincident with a decrease in YFP fluorescence. (C) CFP fluorescence of ROIs measured in 7 different cells before (open bars) and after (filled bars) acceptor photobleaching. "Ave" mean ± s.e (n = 7) of CFP fluorescence. *, p < 0.05 after versus before, non parametric paired T-test.
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Figure 4: Sumoylation of C/EBPβ in astrocytes. Primary rat astrocytes were co-transfected with YFP-SUMO-1 and CFP-C/EBPβ expression plasmids. After 24 hr the cells were fixed, and cells co-expressing YFP-SUMO-1 and CFP-C/EBPβ were subjected to FRET analysis. (A) Representative image of CFP and YFP fluorescence after linear un-mixing of emission spectra and merged image showing the region of interest (ROI) analyzed. (B) FRET data after acceptor photobleaching of the ROI indicated ("1") in panel A, showing an increase in CFP fluorescence coincident with a decrease in YFP fluorescence. (C) CFP fluorescence of ROIs measured in 7 different cells before (open bars) and after (filled bars) acceptor photobleaching. "Ave" mean ± s.e (n = 7) of CFP fluorescence. *, p < 0.05 after versus before, non parametric paired T-test.

Mentions: Group data shown in figures 1 and 3 were analyzed by unpaired T-tests; QPCR data at different ages shown in figure 2 was analyzed by 1 way ANOVA and Bonferroni multiple comparison post hoc analyses; FRET data shown in figure 4 was analyzed by non-parametric paired T-test of CFP fluorescence measured before and after photobleaching in the same ROI. Significance was taken at p values < 0.05.


Modulation of inducible nitric oxide synthase expression by sumoylation.

Akar CA, Feinstein DL - J Neuroinflammation (2009)

Sumoylation of C/EBPβ in astrocytes. Primary rat astrocytes were co-transfected with YFP-SUMO-1 and CFP-C/EBPβ expression plasmids. After 24 hr the cells were fixed, and cells co-expressing YFP-SUMO-1 and CFP-C/EBPβ were subjected to FRET analysis. (A) Representative image of CFP and YFP fluorescence after linear un-mixing of emission spectra and merged image showing the region of interest (ROI) analyzed. (B) FRET data after acceptor photobleaching of the ROI indicated ("1") in panel A, showing an increase in CFP fluorescence coincident with a decrease in YFP fluorescence. (C) CFP fluorescence of ROIs measured in 7 different cells before (open bars) and after (filled bars) acceptor photobleaching. "Ave" mean ± s.e (n = 7) of CFP fluorescence. *, p < 0.05 after versus before, non parametric paired T-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667488&req=5

Figure 4: Sumoylation of C/EBPβ in astrocytes. Primary rat astrocytes were co-transfected with YFP-SUMO-1 and CFP-C/EBPβ expression plasmids. After 24 hr the cells were fixed, and cells co-expressing YFP-SUMO-1 and CFP-C/EBPβ were subjected to FRET analysis. (A) Representative image of CFP and YFP fluorescence after linear un-mixing of emission spectra and merged image showing the region of interest (ROI) analyzed. (B) FRET data after acceptor photobleaching of the ROI indicated ("1") in panel A, showing an increase in CFP fluorescence coincident with a decrease in YFP fluorescence. (C) CFP fluorescence of ROIs measured in 7 different cells before (open bars) and after (filled bars) acceptor photobleaching. "Ave" mean ± s.e (n = 7) of CFP fluorescence. *, p < 0.05 after versus before, non parametric paired T-test.
Mentions: Group data shown in figures 1 and 3 were analyzed by unpaired T-tests; QPCR data at different ages shown in figure 2 was analyzed by 1 way ANOVA and Bonferroni multiple comparison post hoc analyses; FRET data shown in figure 4 was analyzed by non-parametric paired T-test of CFP fluorescence measured before and after photobleaching in the same ROI. Significance was taken at p values < 0.05.

Bottom Line: 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.Our results demonstrate that SUMOylation regulates NOS2 expression in astrocytes, and point to modification of C/EBPbeta as a possible mechanism of action.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, University of Illinois, Chicago, IL 60612, USA. candan@uic.edu

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus