The use of oral contraceptive agents containing synthetic estrogens has been associated with an increased risk of developing hemostatic and thromboembolic abnormalities. These coagulation defects have been attributed to an increased synthesis of clotting factors VIII, IX and XII as well as to a decreased antithrombin activity. Hageman Factor XII (FXII) plays a multifunctional role in blood coagulation, being at the cross-point between the coagulation and fibrinolytic enzymatic cascades. The mechanism by which estrogens regulate FXII gene expression is therefore crucial in understanding the pathogenesis of estrogen-induced alterations of hemostasis. For this purpose, FXII gene transcription was investigated by performing "run-off" assays with liver nuclei isolated from ovariectomized rats, treated or not with 17?-estradiol. Liver FXII gene transcription was about six time higher in treated than in untreated animals, indicating that estrogens induce FXII gene expression in vivo. Analysis of the cloned FXII promoter showed an imperfect palindrome GGGCAnnnTGACC at position -43/-31, resembling the consensus estrogen response element (ERE), and four additional upstream 5'-GGTCA-3' half-sites. Therefore chimeric genes containing portions of the FXII promoter fused to the chloramphenicol acetyl transferase (CAT) coding sequence were transiently co-transfected with the estrogen receptor (ER) in culture cells (NIH3T3 and HepG2), either in the presence or absence of *-estradiol (10-7 M). As a positive control, a similar chimeric plasmid with the promoter of Xenopus vitellogenin gene, a well-known estrogen-responsive gene, fused to CAT, was used in parallel experiments. A 230 bp fragment of FXII promoter (-181/+49) conferred a strong estrogen responsiveness (about 15-fold) to the CAT-reporter gene, confirming that a functional ERE resides in this region. The estrogen effect was highly specific since cognate receptors, such as those for thyroid hormone or retinoic acid, did not stimulate CAT activity. Gel mobility and DNase I footprinting assays demonstrated a specific interaction between, the FXII promoter and estrogen receptor in a region encompassing the palindromic FXII-ERE. Insertion of the putative FXII-ERE into the heterologous thymidine kinase promoter conferred a strong estrogen responsiveness. These results represent not only the first demonstration, at molecular level, of the regulation of a blood coagulation factor gene by estrogens but also the first identification of a functional ERE within this class of genes.
MOLECULAR BASIS OF ESTROGEN REGULATION OF BLOOD COAGULATION FACTOR XII GENE EXPRESSION.
A Farsetti;
1995
Abstract
The use of oral contraceptive agents containing synthetic estrogens has been associated with an increased risk of developing hemostatic and thromboembolic abnormalities. These coagulation defects have been attributed to an increased synthesis of clotting factors VIII, IX and XII as well as to a decreased antithrombin activity. Hageman Factor XII (FXII) plays a multifunctional role in blood coagulation, being at the cross-point between the coagulation and fibrinolytic enzymatic cascades. The mechanism by which estrogens regulate FXII gene expression is therefore crucial in understanding the pathogenesis of estrogen-induced alterations of hemostasis. For this purpose, FXII gene transcription was investigated by performing "run-off" assays with liver nuclei isolated from ovariectomized rats, treated or not with 17?-estradiol. Liver FXII gene transcription was about six time higher in treated than in untreated animals, indicating that estrogens induce FXII gene expression in vivo. Analysis of the cloned FXII promoter showed an imperfect palindrome GGGCAnnnTGACC at position -43/-31, resembling the consensus estrogen response element (ERE), and four additional upstream 5'-GGTCA-3' half-sites. Therefore chimeric genes containing portions of the FXII promoter fused to the chloramphenicol acetyl transferase (CAT) coding sequence were transiently co-transfected with the estrogen receptor (ER) in culture cells (NIH3T3 and HepG2), either in the presence or absence of *-estradiol (10-7 M). As a positive control, a similar chimeric plasmid with the promoter of Xenopus vitellogenin gene, a well-known estrogen-responsive gene, fused to CAT, was used in parallel experiments. A 230 bp fragment of FXII promoter (-181/+49) conferred a strong estrogen responsiveness (about 15-fold) to the CAT-reporter gene, confirming that a functional ERE resides in this region. The estrogen effect was highly specific since cognate receptors, such as those for thyroid hormone or retinoic acid, did not stimulate CAT activity. Gel mobility and DNase I footprinting assays demonstrated a specific interaction between, the FXII promoter and estrogen receptor in a region encompassing the palindromic FXII-ERE. Insertion of the putative FXII-ERE into the heterologous thymidine kinase promoter conferred a strong estrogen responsiveness. These results represent not only the first demonstration, at molecular level, of the regulation of a blood coagulation factor gene by estrogens but also the first identification of a functional ERE within this class of genes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
