mitopodozide and Hyperglycemia

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.

Topics: Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Glomerular Mesangium; Glucose; Humans; Hyperglycemia; Imidazoles; Indoles; Maleimides; Mitogen-Activated Protein Kinases; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Podophyllin; Podophyllotoxin; Promoter Regions, Genetic; Pyridines; RNA, Messenger; Sp1 Transcription Factor; Tetradecanoylphorbol Acetate; Tissue Distribution; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1