Factor XII (FXII) is a liver-specific zymogen involved in the regulation of hemostasis, particularly in the activation of fibrinolysis. Transcription of the FXII gene is stimulated by estrogens through specific interaction of the estrogen receptor α (erα) with an estrogen response element present on FXII promoter. Interestingly, the magnitude of Erα induction in liver Hepg2 cells is much lower than in NIH3T3 fibroblasts, suggesting that cell- specific factors may modulate Erα-dependent trans-activation. Comparative footprinting analysis of FXII promoter (from nucleotides -181 to +49) in liver vs. non-liver cell environments allowed identification of four deoxyribonuclease I-protected sites only in the presence of Hepg2 nuclear extracts. Computerized homology search identified sites III and IV as consensus binding sequences for the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4), formerly an orphan receptor belonging to the superfamily of steroid/thyroid hormone nuclear receptors. In transient transfection assays in NIH3T3 cells, HNF-4 significantly inhibited (70%) estrogen induction of FXII promoter while not affecting basal promoter activity. Conversely, HNF-4 did not inhibit estrogen inducibility of FXII promoter in Hepg2 cells due to the high endogenous levels of HNF-4 protein. In gel shift assays, HNF-4, either present in Hepg2 nuclear extracts or generated by in vitro transcription/translation, specifically bound FXII promoter. This interaction is strictly required in eliciting the antagonistic effect because in NIH3T3 cells, selective mutations of sites III and IV abrogated HNF-4 inhibitory properties. In the liver-specific environment, the same mutant construct exhibited higher estrogen-dependent inducibility compared with native promoter. Rescue of estrogen responsiveness was also achieved using a dominant negative HNF-4, which counteracted endogenous HNF- 4 activity. In conclusion, our findings address a direct role for HNF-4 in modulating estrogen-dependent transcription of the FXII gene promoter.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism