AP-1 proteins mediate hyperglycemia-induced activation of the human TGF-beta 1 promoter in mesangial cells

Hyperglycemia-induced overproduction of the prosclerotic cytokine transform ing growth factor-beta1 (TGF-beta1) has been implicated in the pathogenesis of diabetic nephropathy. Because high glucose and phorbol esters (PMA) inc rease TGF-PI mRNA levels in mesangial cells, this study was designed to cha racterize 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 fo und that 30 mM glucose induced a nearly twofold increase in TGF-beta1 promo ter activity after 24 h of incubation in human and porcine mesangial cells. Stimulation by PMA was more effective (2.3-fold). Mutagenesis in either on e of the two or both activating protein-1 (AP-1) binding sites abolished th e high glucose and the PMA effect. Furthermore, addition of the AP-1 inhibi tor curcumin obliterated the glucose response. Corresponding experiments re vealed that the transcription factor stimulating protein 1 was not involved in mediating the glucose effect. The high glucose-induced TGF-beta1 promot er 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 protein s of mesangial cells, particularly to box B, Supershift assays demonstrated that JunD and c-Fos were present in the protein-DNA complexes under contro l 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 a nd p38 mitogen-activated proteinkinase in hyperglycemia-induced TGF-beta1 g ene expression.