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.
Abdul-Hafez
Amal
A
Department of Physiology, Michigan State University, East Lansing, MI 48824, USA.
Shu
Ruijie
R
Uhal
Bruce D
BD
D000808:Angiotensinogen / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003609:Dactinomycin / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000166:cytology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun; D020871:RNA Stability; D013552:Swine; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*
D051793:Hypoxia-Inducible Factor 1; D011500:Protein Synthesis Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D053773:Transforming Growth Factor beta1; D000808:Angiotensinogen; D003609:Dactinomycin