Factor XII (FXII) is a liver-specific zymogen acting at the crosspoint between the coagulation and fibrinolytic enzymatic cascades. However, FXII appears more relevant in fibrinolysis since hereditary FXII deficiency produces a thrombophilic state, clinically characterized by venous thrombosis. We have previously demonstrated that estrogens stimulate FXII gene expression, both in vivo and in vitro, predominantly at the level of transcription. Estrogen induction of FXII gene expression may contribute to determine the cardiovascular protective effect associated to estrogen replacement therapy in post-menopausal women. Interestingly, we found that estrogen induction of FXII gene transcription is significantly lower in human epatoma (HepG2) as compared to NIH3T3 cells (5- vs. 15-fold, respectively), suggesting that cell-specific factors may modulate FXII estrogen responsiveness. With the aim of identifying these factors, Dnase I footprint assays were performed using HepG2 nuclear extracts. Four protein binding sites were identified within FXII promoter region (nt -181/+49) which encompasses the transcription start site. Site I (nt -44/-31) corresponds to the previously characterized estrogen response element (ERE), which mediates functional interaction with the estrogen receptor (ER). Site II (nt -26/-19) exhibits sequence similarity to TATA-like motifs while site III contains a Dnase I hypersensitive site. Computerized homology search identified site IV as the consensus sequence for binding of the liver-enriched transcription factor HNF-4, an orphan receptor belonging to the superfamily of steroid/thyroid hormone nuclear receptors and required for tissue-specific expression of liver-restricted genes. Transient transfection assays in NIH3T3 cells, in the presence of ER and HNF-4, resulted in a significant inhibition (~ 75%) of the estrogen induction of FXII. Conversely, basal activity of FXII promoter was not affected by HNF-4 alone. These results suggest that HNF-4 is able to interfere with ER transcriptional activity. Interestingly, no inhibition of ER-dependent FXII induction was elicited in HepG2 cells which, by expressing high levels of endogenous HNF-4 protein, already exhibit maximal inhibition of estrogen stimulation of FXII gene. Interaction of the FXII putative HNF-4 binding site (site IV) with HNF-4 protein, generated in an in vitro transcription/translation system, produced a retarded complex in electrophoretic mobility shift assays, indicating that physical interaction between the orphan receptor and FXII promoter is required in eliciting the antagonistic effect. The specificity of the protein-DNA complex was confirmed by co-migration of a similar complex formed between HNF-4 and an oligonucleotide spanning the ?1-antitrypsin HNF-4-specific binding site. In conclusion, our findings indicate that multiple interactions between different, hormone-dependent and tissue-specific transcription factors are required to ensure optimal control of FXII gene expression, thus contributing to the maintenance of the physiological balance between circulating pro- and anti-coagulants.
Orphan receptor hepatocyte nuclear factor 4 (HNF-4) antagonizes estrogen receptor-mediated induction of blood coagulation factor XII
A Farsetti;F Moretti;
1997
Abstract
Factor XII (FXII) is a liver-specific zymogen acting at the crosspoint between the coagulation and fibrinolytic enzymatic cascades. However, FXII appears more relevant in fibrinolysis since hereditary FXII deficiency produces a thrombophilic state, clinically characterized by venous thrombosis. We have previously demonstrated that estrogens stimulate FXII gene expression, both in vivo and in vitro, predominantly at the level of transcription. Estrogen induction of FXII gene expression may contribute to determine the cardiovascular protective effect associated to estrogen replacement therapy in post-menopausal women. Interestingly, we found that estrogen induction of FXII gene transcription is significantly lower in human epatoma (HepG2) as compared to NIH3T3 cells (5- vs. 15-fold, respectively), suggesting that cell-specific factors may modulate FXII estrogen responsiveness. With the aim of identifying these factors, Dnase I footprint assays were performed using HepG2 nuclear extracts. Four protein binding sites were identified within FXII promoter region (nt -181/+49) which encompasses the transcription start site. Site I (nt -44/-31) corresponds to the previously characterized estrogen response element (ERE), which mediates functional interaction with the estrogen receptor (ER). Site II (nt -26/-19) exhibits sequence similarity to TATA-like motifs while site III contains a Dnase I hypersensitive site. Computerized homology search identified site IV as the consensus sequence for binding of the liver-enriched transcription factor HNF-4, an orphan receptor belonging to the superfamily of steroid/thyroid hormone nuclear receptors and required for tissue-specific expression of liver-restricted genes. Transient transfection assays in NIH3T3 cells, in the presence of ER and HNF-4, resulted in a significant inhibition (~ 75%) of the estrogen induction of FXII. Conversely, basal activity of FXII promoter was not affected by HNF-4 alone. These results suggest that HNF-4 is able to interfere with ER transcriptional activity. Interestingly, no inhibition of ER-dependent FXII induction was elicited in HepG2 cells which, by expressing high levels of endogenous HNF-4 protein, already exhibit maximal inhibition of estrogen stimulation of FXII gene. Interaction of the FXII putative HNF-4 binding site (site IV) with HNF-4 protein, generated in an in vitro transcription/translation system, produced a retarded complex in electrophoretic mobility shift assays, indicating that physical interaction between the orphan receptor and FXII promoter is required in eliciting the antagonistic effect. The specificity of the protein-DNA complex was confirmed by co-migration of a similar complex formed between HNF-4 and an oligonucleotide spanning the ?1-antitrypsin HNF-4-specific binding site. In conclusion, our findings indicate that multiple interactions between different, hormone-dependent and tissue-specific transcription factors are required to ensure optimal control of FXII gene expression, thus contributing to the maintenance of the physiological balance between circulating pro- and anti-coagulants.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
