Molecular Cloning and Analysis of the Rat Inducible Nitric Oxide Synthase Gene Promoter in Aortic Smooth Muscle Cells

doi:10.1016/S0006-2952(98)00078-1

Biochemical Pharmacology

Volume 55, Issue 11, 1 June 1998, Pages 1873-1880

https://doi.org/10.1016/S0006-2952(98)00078-1 Get rights and content

Abstract

We have cloned five DNA fragments (−0.32, −0.48, −1.7, −3.2, and −5.1 kb) of the 5′-flanking region of the rat inducible nitric oxide synthase (iNOS) gene from rat genomic DNA. The functional importance of the 5′-flanking region was determined by transient expression of iNOS promoter-luciferase constructs in cultures of rat aortic smooth muscle cells. The −0.48 kb construct, containing one nuclear factor κB (NF-κB) binding site, expressed basal promoter activity but showed only a 1.5- and 1.7-fold increase in luciferase activity in response to lipopolysaccharide (LPS) or a cytokine mixture, respectively. However, the −3.2 kb construct (containing a second NF-κB binding site) showed full promoter activity with a 24-fold increase in response to LPS or cytokine mixture. The −5.1 kb construct showed no further increase in luciferase activity, suggesting that the 1.9 kb upstream of −3.2 kb may not be important in rat iNOS regulation. Rat iNOS promoter induction did not appear to be transcriptionally regulated by NO since NOS inhibitors did not affect induction. These data are in marked contrast to the mouse iNOS promoter in which a DNA sequence as short as a −85 bp, containing one NF-κB site, confers 10-fold inducibility by LPS. The present findings demonstrate that the rat iNOS gene is transcriptionally regulated by cytokines and LPS, but, unlike the mouse gene, the downstream NF-κB site does not appear to be a key region in responses to cytokines and LPS. These data suggest that the regulation of the rat gene may require the coexistence of at least two NF-κB sites or other elements upstream of −0.48 kb of the 5′-flanking region.

Section snippets

Reagents

Human recombinant IL-1β was obtained from Boehringer. TNF-α was purchased from R & D Systems. LPS, l-NMMA and l-NAME were purchased from Sigma. Tissue culture media and Lipofectamine were obtained from Life Technologies.

Cell Culture

RASMC were harvested from Wistar rats (Harlan) by enzymatic dissociation, using standard methods [20]. The cells were identified as smooth muscle by indirect immunofluorescent staining for α-actin, using mouse anti-α-actin antibody and anti-mouse IgG FITC conjugate. The positive

Cloning, Sequence, and Analysis of the 5′-Flanking Region of the Rat iNOS

Four DNA fragments with 0.404, 0.572, 3.28, and 5.2 kb of the 5′-flanking region of the rat iNOS were obtained by polymerase chain reaction amplification from PvuII, SspI, DraI, and ScaI DNA pools, respectively. The EcoRV DNA pool was not amplified. All DNA fragments were cloned into a luciferase containing vector, PGL3-Basic. The DNA sequences of the −3.2-kb (−3196 to +88) construct, as well as of the 404 (−316 to +88) and 572 (−484 to +88) bp, were determined by dye terminator chemistry on

Discussion

Type II NOS can be induced in many species and cell types, and the molecular regulation of this gene appears to differ among cell types. For example, in a mouse macrophage cell line, RAW 264.7, the promoter region that responds to LPS contains only a single downstream NF-κB binding site [11]. However, in the VSMC cell line A7r5, it is the −890-to −1002-bp region (also containing another NF-κB site) that best responds to stimulation by cytokines [18]. Still, in RASMC it is the −234 to +31 region

Acknowledgements

This work was supported by Grant HL52958. We thank Dr. Richard C. Venema for helpful discussion and suggestions and Ms. Aretha Bogan for preparing the manuscript.

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We investigated the regulatory effect of glucosamine (GlcN) for the production of nitric oxide (NO) and expression of inducible NO synthase (iNOS) under various glucose conditions in macrophage cells. At normal glucose concentrations, GlcN dose dependently increased LPS-stimulated production of NO/iNOS. However, GlcN suppressed NO/iNOS production under high glucose culture conditions. Moreover, GlcN suppressed LPS-induced up-regulation of COX-2, IL-6, and TNF-α mRNAs under 25 mm glucose conditions yet did not inhibit up-regulation under 5 mm glucose conditions. Glucose itself dose dependently increased LPS-induced iNOS expression. LPS-induced MAPK and IκB-α phosphorylation did not significantly differ at normal and high glucose conditions. The activity of LPS-induced nuclear factor-κB (NF-κB) and DNA binding of c-Rel to the iNOS promoter were inhibited under high glucose conditions in comparison with no significant changes under normal glucose conditions. In addition, we found that the LPS-induced increase in O-GlcNAcylation as well as DNA binding of c-Rel to the iNOS promoter were further increased by GlcN under normal glucose conditions. However, both O-GlcNAcylation and DNA binding of c-Rel decreased under high glucose conditions. The NF-κB inhibitor, pyrrolidine dithiocarbamate, inhibited LPS-induced iNOS expression under high glucose conditions but it did not influence iNOS induction under normal glucose conditions. In addition, pyrrolidine dithiocarbamate inhibited NF-κB DNA binding and c-Rel O-GlcNAcylation only under high glucose conditions. By blocking transcription with actinomycin D, we found that stability of LPS-induced iNOS mRNA was increased by GlcN under normal glucose conditions. These results suggest that GlcN regulates inflammation by sensing energy states of normal and fuel excess.
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Nitric oxide (NO) is an important signaling molecular in the immune system of all vertebrates and invertebrates for pathologic and physiologic process, and it is largely produced by inducible nitric oxide synthase (iNOS). To uncover key mechanisms regulating NOS expression in sea cucumber Apostichopus japonicus, we amplified a fragment of the NOS promoter by genome walking approach and characterized putative transcription factor binding motifs using luciferase assay. Transient transfection of EPC cells using 5′-deletion constructs linked to luciferase reporter revealed that the region −614/+39 contributed importantly to expression of the AjNOS gene, and the −614 bp of the 5′-flanking region of the AjNOS gene responded well to LPS. Analysis of the functional promoter region revealed the presence of two potential NF-κB (−375 bp to −366 bp, −76 bp to −67 bp) and three STAT binding sites (−284 bp to −276 bp, −95 bp to 87 bp, −81 bp to −73 bp). When luciferase reporter vector and expression vector co-transfected revealed that NF-κB/Rel, but not STAT5, activate the AjNOS promoter fragment. Furthermore, two truncated reporter vectors co-transfected with vector expressing NF-κB/Rel revealed that the first NF-κB binding site (−375 bp to −366 bp) was essential for the ability of this promoter to induce AjNOS transcription. In addition, blocking the AjRel by SN50 (NF-κB inhibitory peptide) depressed the AjNOS expression and NO production both in vivo and in vitro, respectively, revealing that AjRel might directly modulate AjNOS. All our findings confirmed that NF-κB dependent mechanisms regulating expression of AjNOS and suggested a means of linking NO production to the immune response.
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Citation Excerpt :
All mammalian iNOS promoters possess binding sites for the transcription factor NF-κB near the TATA box. The mammalian promoters also display binding sites at position −900 for transcription factors induced by IFN-γ [6,36,38]. A 50-fold higher induction was seen when 8.3 kb fragment of iNOS promoter is used where the transcription sites seem to be located upstream of −3.8 kb of 5′ region of human iNOS gene [6,12,22].
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Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stimulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA were performed on the infected/stimulated A549 cells.
Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimulated with IFN-γ or a combination of three cytokines (IFN-γ, TNF-α and IL-1β) formed DNA protein complexes with probes from both −5.2 kb region (specific for binding of STAT-1 protein) and −5.8 kb region (specific for binding of both STAT-1 and NF-κB) of the iNOS promoter. However, TNF-α or IL-1β stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from −5.2 kb region.
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RNA interference-produced autoregulation of inducible nitric oxide synthase expression
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Citation Excerpt :
As shown in Fig. 1, all six shRNAs significantly inhibited the rat iNOS expression. Since the 3.2 kb iNOS promoter was found to be fully functional in RASMCs [14], we used these cells for the majority of our study. LPS induced a dose-dependent increase of shRNA4 in the RASMCs transfected with piNOS-shRNA4.
Vector-mediated delivery of short-hairpin RNA (shRNA) to regulate gene expression holds a great therapeutic promise. We hypothesize that gene expression can be autoregulated with RNA interference. We used inducible nitric oxide synthase (iNOS) as a gene model to test this hypothesis. Lipopolysaccharide dose-dependently increased iNOS in rat aortic smooth muscle cells and the nitrite production from these cells. These increases were attenuated in cells transfected with plasmids containing code for iNOS shRNA whose expression was controlled by an iNOS promoter. The production of shRNA was lipopolysaccharide dose-dependent. The lipopolysaccharide-induced iNOS expression in rat C6 glioma cells also was attenuated by transfection with plasmids containing the iNOS shRNA code. These results provide proof-of-concept evidence for using RNA interference technique to achieve autoregulation of gene expression.

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¹: The nucleotide sequence data reported in this paper have been submitted to the GenBank with the accession number AF042085.

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Original ArticlesMolecular Cloning and Analysis of the Rat Inducible Nitric Oxide Synthase Gene Promoter in Aortic Smooth Muscle Cells 1

Abstract

Section snippets

Reagents

Cell Culture

Cloning, Sequence, and Analysis of the 5′-Flanking Region of the Rat iNOS

Discussion

Acknowledgements

Cell

Biochem Biophys Res Commun

J Biol Chem

Mol Pharmacol

Biochem Biophys Res Commun

Cell

J Biol Chem

Biochim Biophys Acta

Biochim Biophys Acta

Anal Biochem

Biochem Biophys Res Commun

Biochem Biophys Res Commun

J Biol Chem

NO at work

Cell

Nitric oxidePhysiology, pathophysiology, and pharmacology

Pharmacol Rev

Original Articles
Molecular Cloning and Analysis of the Rat Inducible Nitric Oxide Synthase Gene Promoter in Aortic Smooth Muscle Cells ¹