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Regulation of the murine inducible nitric oxide synthase gene by dexamethasone involves a heterogeneous nuclear ribonucleoprotein I (hnRNPI) dependent pathway
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2007 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 44, no 12, 3204-3210 p.Article in journal (Refereed) Published
Abstract [en]

Glucocorticoids down regulate the inducible nitric oxide synthase (iNOS) gene both transcriptionally and post-transcriptionally. The posttranscriptional events are suggested to involve destabilization of the iNOS transcript although the molecular mechanisms for this effect are not known. Recently, our laboratory demonstrated a lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-induction-dependent interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) I and hnRNPL with a destabilizing element contained in the Tuntranslated region (UTR) of iNOS mRNA. The aim of this study was to investigate if dexamethasone, which down regulates iNOS, is able to modulate this protein-mRNA interaction. As expected, dexamethasone inhibited the induction of iNOS by LPS and IFN-gamma in RAW 264.7 cells, and destabilized the iNOS mRNA. Dexamethasone also counteracted the LPS/IFN gamma-induced disappearance of a gel shifted iNOS mRNA-protein complex containing hnRNPI and hnRNPL. UV cross-linking and Western blot analyses revealed that the RNA-binding and levels of hnRNPI, which decreased by LPS/IFN gamma treatment, were restored by dexamethasone. The results support our hypothesis that hnRNPI is pivotal in the post-transcriptional regulation of iNOS and strongly suggest that hnRNPI is one of the trans-acting factors mediating the post-transcriptional effects of dexamethasone.

Place, publisher, year, edition, pages
2007. Vol. 44, no 12, 3204-3210 p.
National Category
Biological Sciences Chemical Sciences Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-93761DOI: 10.1016/j.molimm.2007.01.029ISI: 000246921500015OAI: oai:DiVA.org:uu-93761DiVA: diva2:167340
Available from: 2005-11-25 Created: 2005-11-25 Last updated: 2011-02-08Bibliographically approved
In thesis
1. Post-Transcriptional Regulation of the Murine Inducible Nitric Oxide Synthase Gene
Open this publication in new window or tab >>Post-Transcriptional Regulation of the Murine Inducible Nitric Oxide Synthase Gene
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented.

Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA.

Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. Trans-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs.

In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 101 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 22
Pharmaceutical biochemistry, iNOS, inducible nitric oxide synthase, gene regulation, post-transcriptional, mRNA stability, RNA-protein interactions, dexamethasone, murine, heterogeneous nuclear ribonucleoprotein, hnRNPI, hnRNPL, Farmaceutisk biokemi
National Category
Pharmaceutical Sciences
urn:nbn:se:uu:diva-6137 (URN)91-554-6402-5 (ISBN)
Public defence
2005-12-16, Room C4:305, BMC, Husargatan 3, UPPSALA, 09:00
Available from: 2005-11-25 Created: 2005-11-25Bibliographically approved

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