Molecular cloning and analysis of the rat inducible nitric oxide synthase gene promoter in aortic smooth muscle cells

Hanfang Zhang, Xunsheng Chen, Xingwu Teng, Connie Snead, John D. Catravas

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)1873-1880
Number of pages8
JournalBiochemical Pharmacology
Volume55
Issue number11
DOIs
StatePublished - Jun 1 1998

Fingerprint

Cloning
Molecular Cloning
Nitric Oxide Synthase Type II
Smooth Muscle Myocytes
Muscle
Rats
Genes
Cells
Lipopolysaccharides
5' Flanking Region
Luciferases
Cytokines
Binding Sites
DNA sequences
DNA
Cell culture

Keywords

  • Cytokines
  • INOS promoter
  • LPS
  • NF-κB
  • Vascular smooth muscle cells

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Cite this

Molecular cloning and analysis of the rat inducible nitric oxide synthase gene promoter in aortic smooth muscle cells. / Zhang, Hanfang; Chen, Xunsheng; Teng, Xingwu; Snead, Connie; Catravas, John D.

In: Biochemical Pharmacology, Vol. 55, No. 11, 01.06.1998, p. 1873-1880.

Research output: Contribution to journalArticle

Zhang, Hanfang ; Chen, Xunsheng ; Teng, Xingwu ; Snead, Connie ; Catravas, John D. / Molecular cloning and analysis of the rat inducible nitric oxide synthase gene promoter in aortic smooth muscle cells. In: Biochemical Pharmacology. 1998 ; Vol. 55, No. 11. pp. 1873-1880.
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AU - Catravas, John D.

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