Nitric Oxide Donors Inhibit Luciferase Expression in a Promoter-independent Fashion

Abstract

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 promoterindependent fashion in both viral and eukaryotic promoters, with a reduction to nearly 50% in the presence of 100 M NO donor. Addition of an SV40 enhancer downstream of the luciferase coding region shifted NO donor inhibition to the right, with inhibition at ϳ300 M. 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. Nitric oxide (NO)1 is a pluripotent regulatory gas with diverse biological effects on numerous enzymes, receptors, structural proteins, and transcriptional factors (1). Studies aimed at understanding the mechanisms by which NO has these myriad actions have represented one of the most rapidly growing areas of research during the last decade. Many reports have shown that NO alters proteins by regulating gene transcription through alterations in promoter specific activity; i.e. NO has been reported to suppress both Egr-1 (2) and PKG-I␣ (3) through control of their respective promoters. A partial list of similarly inhibited gene expression through regulation of promoter activity includes cytokines (4), cytochrome P450 enzymes (5), growth factors (6) as well as secondary inhibition of gene expression stimulatory by factors such as 1␣,25(OH) 2 D 3 (7) and estradiol (8). On the other hand, the ability of nitric oxide to up-regulate promoter activity has been less well reported, although many proteins are increased after exposure to nitric oxide (9 -11). Thus, the ability of nitric oxide to inhibit promoters seems to be widespread.We also found that nitric oxide inhibited the expression of a protein we were interested in; when we turned to transcriptional assays, we also measured effects of the permeable molecule on promoter-driven firefly luciferase. However, while studying a series of promoter deletions, we found that the effect of nitric oxide seemed to be nonspecific. This led us to consider whether our findings might be explained by a more general effect of NO on the luciferase reporter...

La-Related Protein 4 Binds Poly(A), Interacts with the Poly(A)-Binding Protein MLLE Domain via a Variant PAM2w Motif, and Can Promote mRNA Stability

“…This showed that expression of F-LARP4 stabilized luciferase mRNA compared to the vector control, in which luciferase mRNA decayed with a half-life of ϳ4 h (Fig. 9A, vector), in agreement with previous reports (4,23). We conclude that enhanced expression of LARP4 prolonged luciferase mRNA half-life.…”

“…ITC of PAM2w-MLLE interactions was carried out in 50 mM NaH 2 PO 4 , 100 mM NaCl, pH 7.0, with a protein concentration of 170 M for 4.5 mM LARP4 (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) peptide and a protein concentration 85 M for 1.6 mM LARP4 .…”

STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide

“…The pGL3 control vector expressing the luciferase under the control of the SV40 promoter was highly expressed in these cells and there was a 60 % reduction in luciferase activity following DETA/NO treatment (results not shown), as previously reported [37]. Transient transfections of HuH7 cells with the pGL2-IRP1P0.1 construct containing the minimal promoter fragment showed that the basal luciferase activity produced by this construct was very low and treatment of the cells for 48 h with a chemical NO donor (DETA/NO, 250 µM), induced a statistically significant 2-fold reduction (P < 0.05) in luciferase activity ( Figure 1C).…”

Thioredoxin-interacting Protein (Txnip) Gene Expression

“…The mild inhibitory effect of NaN 3 or rotenone on ChoRE-mutated Txnip promoter might be due to residual activities of the mutated ChoREs or, more likely, was not Txnip promoter-specific. For instance, the OXPHOS inhibitor NO has been shown to repress the luciferase expression in a promoter-independent fashion (28). Thus, we conclude that OXPHOS inhibitors repress the Txnip expression at the transcriptional level, which is mediated largely by the ChoREs on the Txnip promoter.…”