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-β (TGF-β)/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-β 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.