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Nitric Oxide Inhibits Endothelial IL-1β-induced ICAM-1 Gene Expression at the Transcriptional Level Decreasing Sp1 and AP-1 Activity

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Abstract

Background

Nitric oxide (NO) has frequently been shown to inhibit leukocyte adherence to activated endothelium thus displaying anti-adhesive and immunosuppressive activities. A molecular mechanism contributing to this effect is described.

Materials and Methods

Primary murine aortic endothelial cells were activated with interleukin (IL)-1β to express intercellular adhesion molecule-1 (ICAM-1) mRNA in the presence or absence of the physiological spontaneous NO-donor S-nitrosocysteine. Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyse the effects of NO on ICAM-1 mRNA expression and on the activity of transcription factors involved in ICAM-1 transcription. In addition, luciferase reporter gene activity of cytokine-activated cells transiently transfected with an ICAM-1 promoter-luciferase construct and cultured in the presence of the slow-releasing NO-donor DETA/NO was determined.

Results

NO at subtoxic concentrations decreases IL-1β-induced endothelial ICAM-1 mRNA expression. This inhibition occurs at the transcriptional level, as NO affects IL-1β-induced ICAM-1 promoter activity in transiently transfected cells. Using gel-shift assays and double-stranded oligonucleotide consensus sequences of the known transcription factor binding sites of the ICAM-1 promoter, Sp1 and AP-1 were identified as transcriptional activators of IL-1β-driven ICAM-1 expression. The DNA binding of both of these transcription factors to specific binding sites of the ICAM-1 promoter was decreased in MAEC exposed to NO.

Conclusions

Our studies indicate that the anti-adhesive effect of NO concentrations equivalent to high-output NO synthesis is mediated, at least in part, by inhibition of ICAM-1 expression via a concerted action of NO on the redox-sensitive transcriptional activators Sp1 and AP-1. This molecular mechanism may contribute to the anti-inflammatory actions of NO synthesized by the inducible NO synthase.

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Acknowledgments

We would like to thank Judith P. Johnson (Institute of Immunology, University of Munich, Germany) for providing the reporter plasmid pIC1014, Peter Zipfel (Hans-Knöll-Institute for Natural Products Research, Jena, Germany) for providing recombinant EGR-1, Carsten Carlberg (Institute for Physiological Chemistry I, University of Dusseldorf, Germany) for generous help with the gel-shift and the reporter gene assays, and Ulla Lammersen for technical help. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Kr 1443/3–2 to KDK).

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  1. Research Group Immunobiology 23.12, MED-Heinrich-Heine-University of Dusseldorf, P.O.Box 10 10 07, D-40001, Dusseldorf, Germany

    Denise Berendji-Grün, Victoria Kolb-Bachofen & Klaus-Dietrich Kröncke

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  1. Denise Berendji-Grün
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  2. Victoria Kolb-Bachofen
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  3. Klaus-Dietrich Kröncke
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Correspondence to Klaus-Dietrich Kröncke.

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Berendji-Grün, D., Kolb-Bachofen, V. & Kröncke, KD. Nitric Oxide Inhibits Endothelial IL-1β-induced ICAM-1 Gene Expression at the Transcriptional Level Decreasing Sp1 and AP-1 Activity. Mol Med 7, 748–754 (2001). https://doi.org/10.1007/BF03401964

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