JunD and HIF-1alpha mediate transcriptional activation of angiotensinogen by TGF-beta1 in human lung fibroblasts.

Earlier work showed that TGF-beta1 potently increases angiotensinogen (AGT) gene mRNA in primary human lung fibroblasts. Here the mechanism of TGF-beta1-induced AGT expression was studied in the IMR90 human lung fibroblast cell line. The increase in AGT mRNA induced by TGF-beta1 was completely blocked by actinomycin-D. TGF-beta1 increased the activity of a full-length human AGT promoter-luciferase reporter (AGT-LUC) but did not alter AGT mRNA half-life. Serial deletion analyses revealed that 67% of TGF-beta-inducible AGT-LUC activity resides in a small domain of the AGT core promoter; this domain contains binding sites for hypoxia-inducible factor (HIF)-1 and activation protein-1 (AP-1) transcription factors. TGF-beta1 increased HIF-1alpha protein abundance and the activity of a hypoxia-responsive element reporter; overexpression of HIF-1 increased basal AGT-LUC activity. Both oligonucleotide pulldown and chromatin immunoprecipitation assays revealed increased binding of JunD and HIF-1alpha to the AGT core promoter in response to TGF-beta1. TGF-beta1-inducible AGT-LUC was reduced by an AP-1 dominant negative or by mutation of the AP-1 site. Knockdown of either JunD or HIF-1alpha individually by siRNA partially reduced AGT-LUC. In contrast, simultaneous knockdown of both JunD and HIF-1alpha completely eliminated TGF-beta1-inducible AGT-LUC activity. These data suggest that TGF-beta1 up-regulates AGT transcription in human lung fibroblasts through a mechanism that requires both JunD and HIF-1alpha binding to the AGT core promoter. They also suggest a molecular mechanism linking hypoxia signaling and fibrogenic stimuli in the lungs.