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AP‐1 complexes mediate oxidized LDL‐induced overproduction of TGF‐β 1 in rat mesangial cells
Author(s) -
Wu Zhaolong,
Zhou Qin,
Lan Yang,
Wang Yuancheng,
Xu Xunhui,
Jin Huiming
Publication year - 2004
Publication title -
cell biochemistry and function
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.933
H-Index - 61
eISSN - 1099-0844
pISSN - 0263-6484
DOI - 10.1002/cbf.1096
Subject(s) - microbiology and biotechnology , transfection , reporter gene , electrophoretic mobility shift assay , mesangial cell , transforming growth factor , chemistry , binding site , promoter , transcription factor , transcription (linguistics) , biology , gene expression , gene , biochemistry , in vitro , linguistics , philosophy
Oxidized Low Density Lipoprotein (Ox‐LDL)‐induced overproduction of the prosclerotic cytokine transforming growth factor‐beta1 (TGF‐β 1 ) has been implicated in the pathogensis of renal fibrosis and sclerosis. Because Ox‐LDL increases TGF‐β 1 mRNA levels in rat mesangial cells, our investigation was designed to characterize these effects on the rat TGF‐β 1 promoter activity. We transfected luciferase reporter gene constructs containing TGF‐β 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‐β 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‐β 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‐β 1 gene expression through AP‐1 binding sites and gives rise to the involvement of protein kinase C in Ox‐LDL‐induced TGF‐β 1 gene expression. Copyright © 2004 John Wiley & Sons, Ltd.