ORPHAN RECEPTOR HEPATOCYTE NUCLEAR FACTOR-4 ANTAGONIZES ESTROGEN-RECEPTOR ALPHA-MEDIATED INDUCTION OF HUMAN COAGULATION-FACTOR XII GENE

Factor XII (FXII) is a liver-specific zymogen involved in the regulati on of hemostasis, particularly in the activation of fibrinolysis. Tran scription of the FXII gene is stimulated by estrogens through specific interaction of the estrogen receptor alpha (ER alpha) with an estroge n response element present on FXII promoter. Interestingly, the magnit ude of ER alpha induction in liver HepG2 cells is much lower than in N IH3T3 fibroblasts, suggesting that cell-specific factors may modulate ER alpha-dependent trans-activation. Comparative footprinting analysis of FXII promoter (from nucleotides -181 to +49) in liver vs. non-live r cell environments allowed identification of four deoxyribonuclease I -protected sites only in the presence of HepG2 nuclear extracts. Compu terized homology search identified sites III and IV as consensus bindi ng sequences for the liver-enriched transcription factor hepatocyte nu clear factor-4 (HNF-4), formerly an orphan receptor belonging to the s uperfamily 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 promo ter activity. Conversely, HNF-4 did not inhibit estrogen inducibility of FXII promoter in HepG2 cells due to the high endogenous levels of H NF-4 protein. In gel shift assays, HNF-4, either present in HepG2 nucl ear extracts or generated by in vitro transcription/translation, speci fically bound FXII promoter. This interaction is strictly required in eliciting the antagonistic effect because in NIH3T3 cells, selective m utations of sites III and IV abrogated HNF-4 inhibitory properties. In the liver-specific environment, the same mutant construct exhibited h igher estrogen-dependent inducibility compared with native promoter. R escue of estrogen responsiveness was also achieved using a dominant ne gative HNF-4, which counteracted endogenous HNF-4 activity. In conclus ion, our findings address a direct role for HNF-4 in modulating estrog en-dependent transcription of the FXII gene promoter.