| Angiotensinogen (AGT) is involved in the rate-limiting step of the reninangiotensin-aldosterone pathway and plays an important role in blood pressure regulation. Naturally-occurring polymorphisms in the angiotensinogen locus have been genetically associated with essential hypertension and preeclampsia. However, the etiologic polymorphism(s) and molecular mechanism(s) underlying this association remain unknown. Herein, 21 angiotensinogen promoters from ethnically diverse individuals were screened for importance of 14 polymorphisms in the context of naturally-occurring haplotypes. Analyses were carried out in human hepatocyte, proximal tubule, astrocyte, and mouse adipocyte cell lines, each of which expressed angiotensinogen endogenously. Polymorphisms with the greatest effect on differential haplotype transcription in these cells were -20A/C and -217G/A. The high-expressing alleles (-20C and -217A) are both relatively common worldwide. Further analyses focused on the role of the -20 polymorphism because of its potentially important role not only in regulating baseline blood pressure but also in regulating diabetes- and/or obesity-related blood pressure. In vitro analyses indicated that USF1/2 preferentially bound the -20C allele, whereas in vivo analyses indicated that USF1 and FRA2 preferentially associated with the -20C allele. Co-transfection studies showed that FRA2, but not FRA1, specifically activated the -20C allele in hepatocytes. Adenoviral shRNAs targeting USF1 or USF2 in human hepatocytes, proximal tubule cells, and astrocytes resulted in cell- and allele-specific attenuation of promoter activity as compared to treatment with adenovirus expressing a control shRNA. Furthermore, adenoviral shRNA-mediated knockdown of USF expression in transgenic mice harboring the -20C allele lowered liver and plasma AGT. The overall conclusion is that USF1 and FRA2 preferentially associate with the -20C allele of AGT to increase transcriptional activation while USF2 may influence AGT expression via an indirect mechanism. |
