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Glycated albumin activates PAI-1 transcription through Smad DNA binding sites in mesangial cells.
Am J Physiol Renal Physiol. 2004 Oct; 287(4):F665-72.AJ

Abstract

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.

Authors+Show Affiliations

Deptartment of Pathology, Seoul National University College of Medicine, Seoul 110-799, Korea. hyunsoon@plaza.snu.ac.krNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15198928

Citation

Lee, Hyun Soon, et al. "Glycated Albumin Activates PAI-1 Transcription Through Smad DNA Binding Sites in Mesangial Cells." American Journal of Physiology. Renal Physiology, vol. 287, no. 4, 2004, pp. F665-72.
Lee HS, Moon KC, Song CY, et al. Glycated albumin activates PAI-1 transcription through Smad DNA binding sites in mesangial cells. Am J Physiol Renal Physiol. 2004;287(4):F665-72.
Lee, H. S., Moon, K. C., Song, C. Y., Kim, B. C., Wang, S., & Hong, H. K. (2004). Glycated albumin activates PAI-1 transcription through Smad DNA binding sites in mesangial cells. American Journal of Physiology. Renal Physiology, 287(4), F665-72.
Lee HS, et al. Glycated Albumin Activates PAI-1 Transcription Through Smad DNA Binding Sites in Mesangial Cells. Am J Physiol Renal Physiol. 2004;287(4):F665-72. PubMed PMID: 15198928.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Glycated albumin activates PAI-1 transcription through Smad DNA binding sites in mesangial cells. AU - Lee,Hyun Soon, AU - Moon,Kyung Chul, AU - Song,Chi Young, AU - Kim,Bong Cho, AU - Wang,Suxia, AU - Hong,Hye Kyoung, Y1 - 2004/06/15/ PY - 2004/6/17/pubmed PY - 2004/10/27/medline PY - 2004/6/17/entrez SP - F665 EP - 72 JF - American journal of physiology. Renal physiology JO - Am J Physiol Renal Physiol VL - 287 IS - 4 N2 - 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. SN - 1931-857X UR - https://www.unboundmedicine.com/medline/citation/15198928/Glycated_albumin_activates_PAI_1_transcription_through_Smad_DNA_binding_sites_in_mesangial_cells_ L2 - https://journals.physiology.org/doi/10.1152/ajprenal.00034.2004?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -