Regulation of the Gene Encoding Thrombin-Activable Fibrinolysis Inhibitor

Disequilibrium between coagulation and fibrinolysis can lead to severe haemostatic disorders such as thrombosis and hemophilia. Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFI may also mediate connections between coagulation and inflammation. Studies have associated high plasma TAFI levels with risk for thrombotic diseases. Interestingly, steroid hormones, such as estrogen and progestogens used in hormone replacement therapy or oral contraceptive preparations, have been shown to affect plasma TAFI levels. Regulation of the expression of the gene encoding TAFI, CBP2, is likely an important determinant of the role of the TAFI pathway in vivo; this concept motivated the investigations described in this thesis. In Chapter 2, the results of my research lead to the identification of key transcription factors regulating CPB2. Specifically, we described the binding of NF-Y and HNF-1 to the CPB2 promoter. NF-Y was shown to be an important factor for the basal CPB2 promoter activity. Binding of HNF-1 is essential for the activity of the promoter and is potentially responsible for the liver specific expression of CPB2. In Chapter 3, we set to investigate the effect of female sex hormone on hepatic expression of CPB2. We demonstrated that the levels of TAFI protein secreted from cultured hepatoma cells (HepG2) are decreased by 17beta-estradiol and progesterone. The change in protein expression was paralleled by decreases in CPB2 mRNA abundance and promoter activity. Deletion analysis of the CPB2 promoter indicated that the genomic effects of estrogen and progesterone are likely mediated via a non-classical mechanism. In Chapter 4, we evaluated the effects of various inflammatory mediators on expression of the gene encoding mouse TAFI (Cpb2). Our results showed that Cpb2 mRNA abundance and promoter activity are up-regulated by inflammatory mediators IL-1beta, IL-6, and TNFalpha. We also showed that TNFalpha mediates its effect via the binding of NFkB. Additionally, our results suggest that TNFalpha promotes the binding of NFkB to the promoter by increasing its translocation to the nucleus. The NFkB site is not conserved between human and mouse and may explained the different responses to inflammation observed in vivo.

ROLE OF CAVEOLIN-3 IN THE MODULATION OF HERG POTASSIUM CHANNEL

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr) that is important for cardiac repolarization. Previously, we have discovered that hERG channels rapidly internalize in low extracellular K+ ([K+]o). In cell culture, this process is driven by the endocytic protein, caveolin-1 (Cav1), which is an integral player in the caveolae-dependant endocytosis pathway. However, in the heart, Caveolin-3 (Cav3) is, in fact, the predominant form in the myocyte, and thus may play a direct role in regulating hERG expression in the heart. Thus, I hypothesize that this reduction of hERG conductance in cardiac myocytes derives from the presence of Cav3, which is integral regulator of hERG homeostasis innately in the heart. To investigate the effect of Cav3 on hERG, I overexpressed Cav3 in human embryonic kidney 293 (HEK-293) cells stably expressing hERG channels. Cav3 overexpression significantly and specifically decreased both the hERG current amplitude and the mature channel expression in normal culture conditions. Co-immunoprecipitation analysis and confocal imaging demonstrated an association between hERG and Cav3 in HEK cells as well as rat and rabbit cardiomyocytes. Mechanistically, I discovered that Cav3 possesses a faster turnover rate compared to Cav1, and can enhance hERG degradation through up-regulating mature channel ubiquitination via the ubiquitin ligase, NEDD4-2. Knockdown of Cav3 in neonatal cardiac myocytes also enhanced hERG expression. My data indicate that Cav3 participates in hERG trafficking, and is an important regulator of hERG channel homeostasis in cardiac myocytes. This information provides a platform for future intervention of the hERG-induced type-2 long QT syndrome (LQTS).