Transcriptional regulation of endothelial nitric-oxide synthase by lysophosphatidylcholine.
We have shown that lysophosphatidylcholine (lyso-PC) increases endothelial nitric-oxide synthase (eNOS) expression at the transcriptional level (Zembowicz, A., Tang, J.-L., and Wu, K. K. (1995) J. Biol. Chem. 270, 17006-17010). To elucidate the mechanism by which lyso-PC increases the eNOS transcription, we identified Sp1 sites at -104 to -90 and PEA3 sites at -40 to -24 as being involved in lyso-PC-induced promoter activity. Site-directed mutagenesis of Sp1 sites resulted in a marked reduction of basal and lyso-PC-induced activity whereas PEA3 site mutation abrogated response to lyso-PC. Band shift assays revealed that lyso-PC augmented Sp1 binding activity. Pretreatment of cells or nuclear extracts with okadaic acid reduced the Sp1 binding activity. Furthermore, okadaic acid treatment abrogated the lyso-PC induced promoter augmentation. Lyso-PC increased the nuclear extract protein phosphatase 2A (PP2A) activity, which was suppressed by okadaic acid treatment. These results suggest that lyso-PC up-regulates eNOS transcription by a PP2A-dependent increase in Sp1 binding activity.
Cieslik
K
K
Vascular Biology Research Center and Division of Hematology, University of Texas-Houston Medical School, Houston, Texas 77030, USA.
Zembowicz
A
A
Tang
J L
JL
Wu
K K
KK
D001483:Base Sequence; D019703:Calcineurin / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000032:analysis; D004730:Endothelium, Vascular / Q000187:drug effects*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006801:Humans; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D009687:Nuclear Proteins / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D054648:Protein Phosphatase 2; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D004268:DNA-Binding Proteins; D008244:Lysophosphatidylcholines; D009687:Nuclear Proteins; D014157:Transcription Factors; D019319:Okadaic Acid; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2