AP-1 proteins mediate hyperglycemia-induced activation of the human TGF-beta1 promoter in mesangial cells.
Hyperglycemia-induced overproduction of the prosclerotic cytokine transforming growth factor-beta1 (TGF-beta1) has been implicated in the pathogenesis of diabetic nephropathy. Because high glucose and phorbol esters (PMA) increase TGF-beta1 mRNA levels in mesangial cells, this study was designed to characterize these effects on the human TGF-beta1 promoter activity. With the use of luciferase reporter gene constructs containing TGF-beta1 5'-flanking sequence (from -453 to +11 bp) transfected into mesangial cells, it was found that 30 mM glucose induced a nearly twofold increase in TGF-beta1 promoter activity after 24 h of incubation in human and porcine mesangial cells. Stimulation by PMA was more effective (2.3-fold). Mutagenesis in either one of the two or both activating protein-1 (AP-1) binding sites abolished the high glucose and the PMA effect. Furthermore, addition of the AP-1 inhibitor curcumin obliterated the glucose response. Corresponding experiments revealed that the transcription factor stimulating protein 1 was not involved in mediating the glucose effect. The high glucose-induced TGF-beta1 promoter activation was also prevented by inhibitors of protein kinase C and p38 mitogen-activated proteinkinase. Electrophoretic mobility shift assays with oligonucleotides containing one of the two AP-1 binding sites showed that glucose treatment markedly enhanced the binding activity of nuclear proteins of mesangial cells, particularly to box B. Supershift assays demonstrated that JunD and c-Fos were present in the protein-DNA complexes under control and hyperglycemic conditions. The functional and structural results show that glucose regulates human TGF-beta1 gene expression through two adjacent AP-1 binding sites and gives rise to the involvement of protein kinase C and p38 mitogen-activated protein kinase in hyperglycemia-induced TGF-beta1 gene expression.
Weigert
Cora
C
Department of Internal Medicine, Division of Endocrinology, Metabolism and Pathobiochemistry, University of Tübingen, Tübingen, Germany.
Sauer
Ulrich
U
Institute of Pathology, University of Munich, Munich, Germany.
Brodbeck
Katrin
K
Department of Internal Medicine, Division of Endocrinology, Metabolism and Pathobiochemistry, University of Tübingen, Tübingen, Germany.
Pfeiffer
Andreas
A
Medical Center Hospital Bergmannsheil, Bochum, Germany.
Häring
Hans U
HU
Department of Internal Medicine, Division of Endocrinology, Metabolism and Pathobiochemistry, University of Tübingen, Tübingen, Germany.
Schleicher
Erwin D
ED
Department of Internal Medicine, Division of Endocrinology, Metabolism and Pathobiochemistry, University of Tübingen, Tübingen, Germany.
D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D005920:Glomerular Mesangium / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D006943:Hyperglycemia / Q000235:genetics*; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011033:Podophyllin / Q000031:analogs & derivatives*; D011034:Podophyllotoxin / Q000031:analogs & derivatives; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014018:Tissue Distribution; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000235:genetics*; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases
D004791:Enzyme Inhibitors; D007093:Imidazoles; D007211:Indoles; D008301:Maleimides; D009687:Nuclear Proteins; D011725:Pyridines; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; C508960:TGFB1 protein, human; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C003170:mitopodozide; D011033:Podophyllin; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose; D011034:Podophyllotoxin; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; C093642:SB 203580