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Originally published In Press as doi:10.1074/jbc.M209911200 on January 13, 2003

J. Biol. Chem., Vol. 278, Issue 12, 10232-10238, March 21, 2003
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Nitric Oxide Donors Inhibit Luciferase Expression in a Promoter-independent Fashion*

Xian FanDagger , Eileen Roy, Liping Zhu, Tamara C. Murphy, Mirek Kozlowski, Mark S. Nanes, and Janet Rubin

From the Department of Medicine, Emory University Medical School and Veterans Affairs Medical Center, Atlanta, Georgia 30033

Nitric oxide (NO) is an important molecule with diverse bio-messenger functions including regulation of gene expression. Transcriptional studies using sensitive luciferase reporter systems have suggested that NO inhibits the promoter activity of a variety of genes. Here we report that NO donors (sodium nitroprusside, 2',2'-(hydroxynitrosohydrazono)bis-ethanimine, and (±)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide) decrease luciferase activity in a promoter-independent fashion in both viral and eukaryotic promoters, with a reduction to nearly 50% in the presence of 100 µM NO donor. Addition of an SV40 enhancer downstream of the luciferase coding region shifted NO donor inhibition to the right, with inhibition at ~300 µM. In contrast, when studied in a chloramphenicol acetyltransferase reporter, two promoters indicating inhibition by NO were unaffected. The decrease in luciferase activity was not caused by NO suppression of the luciferase enzyme. Real-time PCR data showed that luciferase mRNA half-life decreased by nearly half in the presence of NO donor (from 75 to 45 min). The SV40 enhancer prolonged luciferase mRNA half-life and somewhat blunted the NO effect. Our data suggest that exogenous NO inhibits luciferase activity in a dose-dependent manner through decreasing luciferase mRNA stability. Thus, the use of luciferase reporter systems to study transcriptional regulation by NO should be attempted with caution.

* This work was supported by Department of Veterans Affairs Merit and Research Enhancement Award Program grants and National Institutes of Health Grant AR42360.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: VAMC-151, Emory University/VA Medical Center, 1670 Clairmont Rd., Decatur, GA 30033. Tel.: 404-321-6111 x6141; Fax: 404-728-7780; E-mail: xfan@emory.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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