Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor-beta 1.
The promoter region of the endothelial cell nitric oxide synthase (ecNOS) gene contains potential response elements for transforming growth factor-beta 1 (TGF beta 1). TGF beta 1 plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy, and angiogenesis. We therefore sought to determine whether TGF beta 1 might modulate ecNOS expression in bovine aortic endothelial cells (BAEC). TGF beta 1 increased ecNOS mRNA in a dose-dependent manner. TGF beta 1 also increased ecNOS protein content. The production of nitrogen oxides (NOx), assessed by chemiluminescence, and nitric oxide synthase activity, assessed by arginine/citrulline conversion were increased in TGF beta 1-treated cells. Transcriptional activity of the 5'-flanking promoter region of the ecNOS gene was increased by TGF beta 1, as assessed by transfection with promoter/luciferase constructs. Deletion analysis suggested that the TGF beta 1-response element was present between nucleotides -1269 and -935 from the first transcription start site, in which a putative nuclear factor-1 (NF-1) binding site existed. Gel shift assays showed that nuclear protein(s), immunologically similar to CCAAT transcription factor/NF-1, bound to the putative NF-1 binding site in a sequence-specific manner. Mutation of the putative NF-1 binding site in the promoter/luciferase construct significantly decreased the responsiveness to TGF beta 1. In conclusion, TGF beta 1 increases ecNOS expression associated with an increase in production of NO in BAEC. This response is probably mediated by transcriptional activation of the ecNOS gene promoter.
Inoue
N
N
Department of Medicine, Emory University School of Medicine, Atlanta, Ga 30322, USA.
Venema
R C
RC
Sayegh
H S
HS
Ohara
Y
Y
Murphy
T J
TJ
Harrison
D G
DG
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051839:NFI Transcription Factors; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C080333:transcription factor nuclear factor 1; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases