Glycated albumin activates PAI-1 transcription through Smad DNA binding sites in mesangial cells.
Amadori-modified glycated albumin stimulates extracellular matrix and transforming growth factor-beta (TGF-beta) expression in cultured mesangial cells. Smad proteins transduce the TGF-beta-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 microg/ml bovine serum albumin (BSA) or glycated BSA (Gly-BSA) for 12-72 h. At 24 h, Gly-BSA stimulated TGF-beta1 and PAI-1 mRNA expression in HMC to 1.8 and 3.2 times that in the BSA-treated control cells. Gly-BSA also activated the PAI-1 promoter luciferase activity 2.3-fold. Gly-BSA-treated cells enhanced Smad2 and Smad3 protein levels 2.5 times the control levels in the nuclei. An 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-fold 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 Gly-BSA-induced PAI-1 mRNA expression. Cotransfection of phosphorothioate CAGA oligonucleotides with PAI-1 reporter vector also blocked 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.
Lee
Hyun Soon
HS
Deptartment of Pathology, Seoul National University College of Medicine, Seoul 110-799, Korea. hyunsoon@plaza.snu.ac.kr
Moon
Kyung Chul
KC
Song
Chi Young
CY
Kim
Bong Cho
BC
Wang
Suxia
S
Hong
Hye Kyoung
HK
D000328:Adult; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003928:Diabetic Nephropathies / Q000378:metabolism / Q000503:physiopathology; D005920:Glomerular Mesangium / Q000166:cytology / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects* / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000235:genetics; D053773:Transforming Growth Factor beta1; D015854:Up-Regulation
D004268:DNA-Binding Proteins; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D012710:Serum Albumin, Bovine