Regulation of the Gene Encoding Human Thrombin-Activable Fibrinolysis Inhibitor by Female Sex Steroids

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme that, once activated by thrombin, the thrombin-thrombomodulin complex, or plasmin, attenuates fibrinolysis. Aberrant regulation of the TAFI pathway alters the balance between coagulation and fibrinolysis and may underlie severe haemostatic disorders such as thrombosis and hemophilia. Indeed, high plasma TAFI levels have been associated with several cardiovascular diseases. It is important to note that there is a large inter-individual variability in plasma TAFI levels within the population, and this variation is primarily due to non-genetic factors. Therefore, variation in TAFI gene expression is a risk factor for thrombotic disorders and may be an important means by which TAFI responds to environmental and physiological stimuli. Novel associations between plasma TAFI levels and sex hormones have triggered interest in determining the role of TAFI as a mediator of the cardioprotective effects of estrogens and progestagens, or as a mediator of the increased thrombotic risk that accompanies use of oral contraceptives or hormone replacement therapy. Plasma TAFI concentrations rise with age in women but not in men, and are elevated in post-menopausal women compared to pre-menopausal women. In addition, plasma TAFI levels have been shown to be decreased by selective estrogen receptor modulators such as HMR 3339 and raloxifene, estradiol plus trimegestone, transdermal estradiol, and oral estradiol plus gestodene. On the other hand, some studies have reported minimal to no change in plasma TAFI levels occurring with the use of oral contraceptive, Raloxifene, or Tamoxifen. Paradoxically, it has been shown that both plasma TAFI levels and clot lysis time rise during pregnancy and then promptly return to basal levels after delivery. These studies illustrate the controversies surrounding the role of sex steroids in modulating plasma TAFI levels. In the present study, we have attempted to directly measure the effect of sex steroids on hepatic TAFI gene expression, and to uncover the molecular mechanisms underlying these regulatory events. HepG2 (human hepatocellular carcinoma) cells were cultured in the presence or absence of progesterone and b-estradiol and TAFI mRNA abundance was measured using real-time RT-PCR. We found that both of these hormones significantly decrease endogenous TAFI mRNA abundance in a dose-dependant manner. To assess the ability of these hormones to influence transcription of the gene encoding TAFI, we treated HepG2 cells that had been transiently transfected with luciferase reporter plasmids containing the 5′-flanking region of the TAFI gene. Interestingly, the change in promoter activity closely paralleled changes in mRNA abundance, suggesting that the effect of the hormones is mediated at the level of transcription. Furthermore, changes in TAFI mRNA abundance following treatments with estrogen were not associated with a decrease in TAFI mRNA stability when compared to the untreated control. TAFI protein levels were also decreased in a dose-dependent manner as assessed by western blot analysis. Inspection of the sequence of the TAFI 5′-flanking region does not show any consensus estrogen responsive elements, although we cannot exclude a role for more complex transcriptional system such as an estrogen response unit. The effect of estrogen could also be performed indirectly through the modulation of other transcription factors such SP-1 or members of the basal transcriptional machinery. We also investigated whether progesterone decreases TAFI gene expression via the binding of the progesterone receptor to the established glucocorticoid responsive element (GRE) within the TAFI promoter. Our results showed that progesterone generates the same decrease in promoter activity even when the GRE site was mutated, indicating that progesterone may act through a different site. In conclusion, our studies are beginning to reveal the molecular basis for the apparent relationship between female sex steroids and plasma concentrations of TAFI: specifically, a direct downregulatory effect on transcription of the gene encoding TAFI.