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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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Presence of C/EBPβ and SUMO-1 on the NOS2 promoter. Primary astrocytes were treated with nothing (C), LPS (L), or LPS+NA (LN) for 4 hours, then ChIP analysis conducted. (A) Two fragments of the rat NOS2 promoter containing the proximal C/EBP-2 (top) or C/EBP-3 (bottom) binding sites were amplified by PCR after immunoprecipitation using (B) anti-C/EBPβ antibody or (C) anti-SUMO-1 antibody alone or followed by anti-C/EBPβ antibody. The products were separated through 2% agarose gels and their accuracy was verified by comparison to input DNA ("in", 5% of total input). The images shown are inverted versions (Adobe Photoshop) of the original gel images and are representative of results obtained in 2 separate experiments. (D) Lysates of astrocytes treated as indicated above were immunoprecipitated (IP) with anti-C/EBPβ antibody and Western blot (W) of the samples was probed with anti-SUMO-1 antibody.
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Figure 5: Presence of C/EBPβ and SUMO-1 on the NOS2 promoter. Primary astrocytes were treated with nothing (C), LPS (L), or LPS+NA (LN) for 4 hours, then ChIP analysis conducted. (A) Two fragments of the rat NOS2 promoter containing the proximal C/EBP-2 (top) or C/EBP-3 (bottom) binding sites were amplified by PCR after immunoprecipitation using (B) anti-C/EBPβ antibody or (C) anti-SUMO-1 antibody alone or followed by anti-C/EBPβ antibody. The products were separated through 2% agarose gels and their accuracy was verified by comparison to input DNA ("in", 5% of total input). The images shown are inverted versions (Adobe Photoshop) of the original gel images and are representative of results obtained in 2 separate experiments. (D) Lysates of astrocytes treated as indicated above were immunoprecipitated (IP) with anti-C/EBPβ antibody and Western blot (W) of the samples was probed with anti-SUMO-1 antibody.

Mentions: The involvement of C/EBPβ and SUMO-1 in NOS2 transcription was further investigated using ChIP assays to detect the association of these proteins with regions of the NOS2 promoter containing either the C/EBP-2 or the C/EBP-3 site (Figure 5A). C/EBPβ was found associated with both the C/EBP-2 and C/EBP-3 sites under all conditions; however binding to C/EBP-2 was increased by treatment with LPS plus NA (Figure 5B). SUMO-1 was detected at both sites, although a stronger signal was obtained with the fragment of the NOS2 promoter containing the C/EBP-3 site (Figure 5C). Treatment with LPS reduced association of SUMO-1 with this site while addition of NA restored it (or prevented that loss). Re-precipitation of SUMO-1 interacting fragments with anti-C/EBPβ antibody showed that under all conditions, the C/EBP-3 site also interacted with C/EBPβ (Figure 5C, bottom panel). In contrast there was little evidence for simultaneous association of both SUMO-1 and C/EBPβ at C/EBP-2 site. An interaction of SUMO-1 with C/EBPβ was confirmed by Western blots which showed that SUMO-1 co-immunoprecipitated with C/EBPβ (Figure 5D), and this interaction was increased by the presence of NA.


Modulation of inducible nitric oxide synthase expression by sumoylation.

Akar CA, Feinstein DL - J Neuroinflammation (2009)

Presence of C/EBPβ and SUMO-1 on the NOS2 promoter. Primary astrocytes were treated with nothing (C), LPS (L), or LPS+NA (LN) for 4 hours, then ChIP analysis conducted. (A) Two fragments of the rat NOS2 promoter containing the proximal C/EBP-2 (top) or C/EBP-3 (bottom) binding sites were amplified by PCR after immunoprecipitation using (B) anti-C/EBPβ antibody or (C) anti-SUMO-1 antibody alone or followed by anti-C/EBPβ antibody. The products were separated through 2% agarose gels and their accuracy was verified by comparison to input DNA ("in", 5% of total input). The images shown are inverted versions (Adobe Photoshop) of the original gel images and are representative of results obtained in 2 separate experiments. (D) Lysates of astrocytes treated as indicated above were immunoprecipitated (IP) with anti-C/EBPβ antibody and Western blot (W) of the samples was probed with anti-SUMO-1 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2667488&req=5

Figure 5: Presence of C/EBPβ and SUMO-1 on the NOS2 promoter. Primary astrocytes were treated with nothing (C), LPS (L), or LPS+NA (LN) for 4 hours, then ChIP analysis conducted. (A) Two fragments of the rat NOS2 promoter containing the proximal C/EBP-2 (top) or C/EBP-3 (bottom) binding sites were amplified by PCR after immunoprecipitation using (B) anti-C/EBPβ antibody or (C) anti-SUMO-1 antibody alone or followed by anti-C/EBPβ antibody. The products were separated through 2% agarose gels and their accuracy was verified by comparison to input DNA ("in", 5% of total input). The images shown are inverted versions (Adobe Photoshop) of the original gel images and are representative of results obtained in 2 separate experiments. (D) Lysates of astrocytes treated as indicated above were immunoprecipitated (IP) with anti-C/EBPβ antibody and Western blot (W) of the samples was probed with anti-SUMO-1 antibody.
Mentions: The involvement of C/EBPβ and SUMO-1 in NOS2 transcription was further investigated using ChIP assays to detect the association of these proteins with regions of the NOS2 promoter containing either the C/EBP-2 or the C/EBP-3 site (Figure 5A). C/EBPβ was found associated with both the C/EBP-2 and C/EBP-3 sites under all conditions; however binding to C/EBP-2 was increased by treatment with LPS plus NA (Figure 5B). SUMO-1 was detected at both sites, although a stronger signal was obtained with the fragment of the NOS2 promoter containing the C/EBP-3 site (Figure 5C). Treatment with LPS reduced association of SUMO-1 with this site while addition of NA restored it (or prevented that loss). Re-precipitation of SUMO-1 interacting fragments with anti-C/EBPβ antibody showed that under all conditions, the C/EBP-3 site also interacted with C/EBPβ (Figure 5C, bottom panel). In contrast there was little evidence for simultaneous association of both SUMO-1 and C/EBPβ at C/EBP-2 site. An interaction of SUMO-1 with C/EBPβ was confirmed by Western blots which showed that SUMO-1 co-immunoprecipitated with C/EBPβ (Figure 5D), and this interaction was increased by the presence of NA.

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