AP-1 complexes mediate oxidized LDL-induced overproduction of TGF-beta(1) in rat mesangial cells.
Oxidized Low Density Lipoprotein (Ox-LDL)-induced overproduction of the prosclerotic cytokine transforming growth factor-beta1 (TGF-beta(1)) has been implicated in the pathogenesis of renal fibrosis and sclerosis. Because Ox-LDL increases TGF-beta(1) mRNA levels in rat mesangial cells, our investigation was designed to characterize these effects on the rat TGF-beta(1) promoter activity. We transfected luciferase reporter gene constructs containing TGF-beta(1) 5'-flanking sequence (from -1550 to +57 bp) into mesangial cells. By assaying progressively deleted mutations in the promoter, we found two regions that were responsible for the induction. One is a negative regulatory region (-422 to -629) which represses the transcription of the TGF-beta(1) gene, the other is a positive regulatory region (-845 to -1550) which enhances the transcription unit efficiently. There is an activating protein-1(AP-1) binding site in the latter region. Mutagenesis in the AP-1 binding sites abolished the Ox-LDL effect. Furthermore, addition of the AP-1 inhibitor curcumin obliterated the Ox-LDL response. The Ox-LDL-induced TGF-beta(1) promoter activation was also prevented by inhibitors of protein kinase C, but not by p38 mitogen-activated protein kinase. Electrophoretic mobility shift assays with oligonucleotides containing AP-1 binding sites showed that Ox-LDL treatment significantly enhanced the binding activity of nuclear proteins of mesangial cells. Supershift assays demonstrated that c-Jun was present in the protein-DNA complexes under stimulation of Ox-LDL. The functional and structural results show that Ox-LDL regulates rat TGF-beta(1) gene expression through AP-1 binding sites and gives rise to the involvement of protein kinase C in Ox-LDL-induced TGF-beta(1) gene expression.
Wu
Zhaolong
Z
Division of Nephrology, Zhongshan Hospital, Shanghai Medical College of Fudan University, Shanghai, P.R. China. professor-wu@yahoo.com.cn
Zhou
Qin
Q
Lan
Yang
Y
Wang
Yuancheng
Y
Xu
Xunhui
X
Jin
Huiming
H
D000818:Animals; D001665:Binding Sites; D003474:Curcumin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008297:Male; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000187:drug effects; D017384:Sequence Deletion; D015398:Signal Transduction / Q000187:drug effects; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis* / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D008077:Lipoproteins, LDL; D009687:Nuclear Proteins; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D053773:Transforming Growth Factor beta1; C099252:oxidized low density lipoprotein; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases; D003474:Curcumin