title: Role of CAGA boxes in the plasminogen activator inhibitor-1 promoter in mediating oxidized low-density lipoprotein-induced transcriptional activation in mesangial cells.
abstract: Oxidized low-density lipoprotein (Ox-LDL) activates transforming growth factor-beta (TGF-beta)/Smad signaling to stimulate plasminogen activator inhibitor-1 (PAI-1) expression in mesangial cells. Smad-binding sequences, termed CAGA boxes, are present in the promoter of human PAI-1 gene, and they mediate TGF-beta transcriptional induction. However, the functional role of each CAGA box in the Ox-LDL-induced PAI-1 promoter activation is unknown. In this study, mutation of 1 of the 3 CAGA boxes located at -730, -580, and -280 of the PAI-1 promoter decreased the Ox-LDL-induced luciferase activity by 40 to 58%, whereas mutations in 2 sites reduced it over 75% or completely abolished it. Overexpression of Smad3 in N-terminal tagged Smad3-transfected cells increased the Ox-LDL-induced transcriptional activation of the PAI-1 promoter, whereas mutation of Smad3 abolished it. Electrophoretic mobility shift assay showed that the labeled -280, -580, and -730 CAGA box probes detected DNA/protein complexes induced by Ox-LDL, whereas mutant probes did not. When nuclear extracts were preincubated with a 100-fold of an unlabeled -280, -580, and -730 CAGA oligonucleotide, the formation of complexes was prevented but not with mutant CAGA box competitors. The addition of anti-Smad3 to the reaction with the labeled -280 or -580 CAGA box probe resulted in a supershift, but not with the -730 CAGA box probe. These results suggest that the 3 CAGA elements in the PAI-1 promoter mediate the Ox-LDL-induced PAI-1 transcription to a different degree, of which the -280 and -580 CAGA regions directly bind to Smad3.
authors.affiliation: Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Bong Cho
authors.initials: BC
authors.lastname: Kim
authors.forename: Chi Young
authors.initials: CY
authors.lastname: Song
authors.forename: Hye Kyoung
authors.initials: HK
authors.lastname: Hong
authors.forename: Hyun Soon
authors.initials: HS
authors.lastname: Lee
mesh_terms: D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D050527:Mesangial Cells / Q000187:drug effects* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015398:Signal Transduction; D051785:Smad Proteins / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D015854:Up-Regulation
chemical_list: D008077:Lipoproteins, LDL; D009841:Oligonucleotides; D017395:Plasminogen Activator Inhibitor 1; D051785:Smad Proteins; C099252:oxidized low density lipoprotein