AP-1 complexes mediate oxidized LDL-induced overproduction of TGF-β1 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 pathogensis 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 OxLDL-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. Copyright (C) 2004 John Wiley Sons, Ltd.