To clarify whether the activating protein-1 (AP-1) is involved in the transcriptional regulation of expression of oxidized low density lipoprotein (Ox-LDL) induced transforming growth factor (TGF)-beta(1) gene and further to investigate the possible signal transduction pathway in the cultured mesangial cells (MsCs).
Firstly, a deletion analysis of SD rat renal mesangial TGF-beta(1) promoter (pRT)--luciferase reporter genes was adopted to identify the possible regulatory regions responding to Ox-LDL. With mutation of AP-1 binding site and addition of the c-Jun/AP-1 inhibitor curcumin, the changes of relative luciferase of pRT stimulated by Ox-LDL were assayed, respectively. Furthermore, the phosphorylation of p38MAPK was detected by Western blotting. Pre-treatment with the different specific kinase inhibitors, the activities of AP-1 induced by Ox-LDL were determined by electrophoretic mobility shift assay (EMSA).
Relative luciferase activity showed two regions responding to stimulation by Ox-LDL in rat TGF-beta(1) promoter: one positive regulatory region (-1 550 to -845) containing an accurate AP-1 binding site, and one negative (-629 to -422) regulatory region. Both mutation of AP-1 binding site and addition of curcumin markedly decrease of activity of TGF-beta(1) promoter. The phosphorylation, but not total protein, of p38MAPK was significantly enhanced by Ox-LDL stimulation. The inhibitor of PKC remarkably reduced the activity of AP-1 induced by Ox-LDL. Reversely, the activity of AP-1 didn't significantly change with the inhibitor of p38MAPK.
In the cultured rat mesangial cells, the AP-1 complex regulates Ox-LDL induced-overproduction of TGF-beta(1) at the transcriptional level. Furthermore, the functional and structural results showed that Ox-LDL regulates rat TGF-beta(1) gene expression through AP-1 binding site and c-Jun/AP-1 complex, it gives rise to the involvement of protein kinase C, but not p38MAPK, in Ox-LDL-induced TGF-beta(1) gene expression.