Amadori-modified glycated albumin stimulates extracellular matrix and transforming growth factor- (TGF-) expression in cultured mesangial cells. Smad proteins transduce TGF--mediated signal, and Smad-binding CAGA sequences are present in the plasminogen activator inhibitor-1 (PAI-1) promoter. This study examined whether glycated albumin induces PAI-1 transcription in human mesangial cells (HMC) through Smad-binding sites in the PAI-1 promoter. Quiescent HMC were exposed to 200 µg/ml bovine serum albumin (BSA) or glycated BSA (Gly-BSA) for 12 to 72 hours. At hour 24, Gly-BSA stimulated TGF-1 and PAI-1 mRNA expression in HMC to 1.8 and 3.2 times the BSA-treated control cells. Gly-BSA also activated the PAI-1 promoter luciferase activity to 2.3-fold. Gly-BSA-treated cells enhanced Smad2 and Smad3 protein levels 2.5 times the control levels in the nuclei. Electrophoretic mobility shift assay performed using CAGA sequences as a probe showed that Gly-BSA increased DNA/protein complexes. When nuclear extracts were preincubated with 100 molar excess of unlabeled CAGA oligonucleotide, the formation of complex was prevented. The DNA binding protein was shown to be Smad3 by antibody supershift. Transfection of phosphorothioate CAGA oligonucleotide, a CAGA antisense analog, inhibited the Gly-BSA-induced PAI-1 mRNA expression. Cotransfection of phosphorothioate CAGA oligonucleotides with PAI-1 reporter vector also blocked the Gly-BSA-induced PAI-1 promoter luciferase activity. These results indicate that Gly-BSA increases DNA binding activity of Smad3 and that it stimulates PAI-1 transcription through Smad-binding CAGA sequences in the PAI-1 promoter in HMC. Thus, progression of diabetic nephropathy may be promoted by PAI-1 upregulation mediated by the glycated albumin-induced Smad/DNA interactions.