TY - JOUR
T1 - Molecular cloning and analysis of the rat inducible nitric oxide synthase gene promoter in aortic smooth muscle cells
AU - Zhang, Hanfang
AU - Chen, Xunsheng
AU - Teng, Xingwu
AU - Snead, Connie
AU - Catravas, John D.
N1 - Funding Information:
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.
PY - 1998/6/1
Y1 - 1998/6/1
N2 - 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.
AB - 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.
KW - Cytokines
KW - INOS promoter
KW - LPS
KW - NF-κB
KW - Vascular smooth muscle cells
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U2 - 10.1016/S0006-2952(98)00078-1
DO - 10.1016/S0006-2952(98)00078-1
M3 - Article
C2 - 9714306
AN - SCOPUS:0032100679
SN - 0006-2952
VL - 55
SP - 1873
EP - 1880
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 11
ER -