| Abstract |
Cardiovascular diseases (CVDs) are the number one cause of death globally. There is unequivocal evidence that high density lipoprotein (HDL) cholesterol levels in plasma are inversely associated with the risk of CVDs. Apolipoprotein E (ApoE) appears to possess a dual function: apart from its role in the clearance of triacylglycerol-rich lipoproteins, it participates in the HDL-sized apoE-containing particles biogenesis. Cholesteryl ester transfer protein (CETP) catalyzes the exchange of triglycerides from apoB-containing particles, such as LDL and VLDL, for cholesteryl esters from the core of HDL particles. Its activity determines the plasma levels of HDL cholesterol. Polymorphisms are observed frequently on the apoE and CETP genes and can significantly influence levels of lipoproteins in the plasma, and the latter gene is considered to be candidate for coronary heart disease. Therefore, in the first chapter of the present thesis, we aimed to identify genetic changes in the human apoE and CETP genes, identified by the group of Dr Anne Tybjaerg-Hansen (Copenhagen University Hospital) in The Copenhagen City Heart Study (CCHS), which may contribute to an up- or down-regulation of the corresponding genes, as well as the establishment of the possible mechanisms responsible for this pattern of regulation. For that purpose we generated and utilized luciferase expression vectors bearing the CETP promoter region or/and the CETP first intron region, as well as luciferase expression vectors bearing the apoE promoter region, bearing either the most common or the less common allele for each SNP and we performed transient transfection assays in human hepatoblastoma (HepG2) cells. We showed that two CETP promoter polymorphisms, -656C>A and -65G>A down-regulate and an up-regulate the promoter activity respectively, when the less common allele is present in each case. Moreover, the first intronic region +191/+1056 of the human CETP gene was shown to significantly down-regulate the promoter’s activity. Importantly it was shown that the combination of the less common -656A allele, of CETP promoter polymorphism -656C>A, with the most common +279G allele, of the CETP first intron polymorphism +279G>A, significantly reduces the activity of the corresponding promoter. In addition, the less common -427C allele, of human apoE promoter polymorphism -427T>C, was shown to down-regulate the apoE promoter activity at a statistically significant degree.
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Hepatocyte nuclear factor 4 alpha (HNF-4α) is a nuclear receptor which activates the expression of a wide variety of genes, especially those involved in intermediary metabolism, including fatty acid, glucose, xenobiotic, cholesterol and drug metabolism, as well as others involved in blood coagulation, urea biosynthesis, hepatitis B infections, and liver differentiation. It appears to be essential both in early as well as in adult development and is mainly expressed in the adult liver, intestine, pancreas, kidney and colon. Mammalian carboxylesterases (CES) comprise a family of genes, whose products are localized in the endoplasmic reticulum of many tissues. These enzymes can efficiently catalyze the hydrolysis of a variety of amide- and ester-containing chemicals, as well as drugs and prodrugs, to the respective free acids. At least 20 mouse Ces genes have been recognized, two of which, carboxylesterase 3 alpha (Ces3a) and carboxylesterase 3 beta (Ces3b), comprise the Ces3-like mouse genes. However, not much is known about carboxylesterase 3 beta (Ces3b) of the mouse, apart from the fact that it is mainly expressed in mouse liver. Preliminary data from our laboratory, using microarrays in HNF-4α liver knock-out mice compared to control mice, revealed a significant number of genes belonging to the family of mouse carboxylesterases, which are up- or down-regulated in the absence of HNF-4α from mouse liver. Among the carboxylesterases down-regulated, Ces3b demonstrated the highest fold change in expression levels. So, the aim of the second chapter of the present thesis was to examine whether mouse Ces3b expression is HNF-4α dependent. To identify potential HNF-4α binding sites in mouse Ces3b promoter, we performed transient transfection assays in human embryonic kidney (HEK) cells, utilizing a series of luciferase reporter plasmids containing consecutive 5’ deletions or a 3’ deletion of the mouse Ces3b promoter along with an expression vector for the cDNA of HNF-4α. We observed that overexpression of HNF-4 significantly up-regulated mouse Ces3b promoter activity in all different Ces3b promoter fragments examined, apart from the shortest fragment bearing the Ces3b promoter region -80 to +4. Importantly, a potential HNF-4α binding site as well as a potential chicken ovalbumin upstream promoter (COUP) binding site, have been identified in the region downstream the +1 transcription start site. It is well known that HNF-4α and COUP transcription factors share common recognition sequence, so perhaps either of these sites could be responsible for the binding of HNF-4α in our transfection assays.
In summary, the findings of the present thesis increase our understanding of the mechanisms that regulate the expression of genes that are involved in lipid and lipoprotein metabolism in the liver and how these mechanisms are dys-regulated by silencing of hepatic nuclear factors or by natural polymorphisms in the promoters or enhancers of the corresponding genes.
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