Role of CAGA boxes in the plasminogen activator inhibitor-1 promoter in mediating oxidized low-density lipoprotein-induced transcriptional activation in mesangial cells.
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.
Kim
Bong Cho
BC
Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
Song
Chi Young
CY
Hong
Hye Kyoung
HK
Lee
Hyun Soon
HS
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
D008077:Lipoproteins, LDL; D009841:Oligonucleotides; D017395:Plasminogen Activator Inhibitor 1; D051785:Smad Proteins; C099252:oxidized low density lipoprotein