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Identifier

000366664

Title

Μηχανισμοί μεταγραφικής ρύθμισης του γονιδίου SR-BI του ανθρώπου

Alternative Title

Transcriptional regulation of the human SR-BI gene.

Author

Μαυρίδου, Σοφία

Thesis advisor

Καρδάσης, Δημήτρης

Reviewer

Τσατσάνης
Ηλιόπουλος
Βενυχάκη
Μπούμπας
Μαργιωρής
Γραβάνης
Ζαννής

Abstract

Scavenger receptor class B type I (SR-BI) facilitates the reverse transport of excess cholesterol from peripheral tissues to the liver via high-density lipoproteins. In steroidogenic tissues, SR-BI supplies cholesterol for steroid hormone production. In the present thesis, we focused on the role of hormone nuclear receptors in human SR-BI gene regulation in hepatic, adrenal and ovarian cells. We show that the transcription of the human SR-BI gene is subject to feedback inhibition by glucocorticoids in adrenal and ovarian cells. SR-BI mRNA levels were increased in adrenals from corticosterone-insufficient mice, whereas corticosterone replacement by oral administration inhibited SR-BI gene expression in these mice. SR-BI mRNA levels were increased in adrenals from wild-type mice treated with metyrapone, a drug that blocks corticosterone synthesis. Experiments in adrenocortical H295R and ovarian SKOV-3 cells using cycloheximide and siRNA-mediated gene silencing revealed that glucocorticoidmediated inhibition of SR-BI gene transcription requires de novo protein synthesis and the glucocorticoid receptor (GR). No direct binding of GR to the SRBI promoter could be demonstrated in vitro and in vivo, suggesting an indirect mechanism of repression of SR-BI gene transcription by GR in adrenal cells. Deletion analysis established that the region of the human SR-BI promoter between nucleotides -201 and -62 is sufficient to mediate repression by 11 glucocorticoids. This region contains putative binding sites for transcriptional repressors that could play a role in SR-BI gene regulation in response to glucocorticoids. In hepatic cells, SR-BI is regulated by an interplay of multiple hepatocytespecific transcription factors such as Hepatocyte Nuclear Factor 3β (HNF-3β or FOXA2) and the orphan nuclear receptor Hepatocyte Nuclear Factor 4 (HNF-4). Using the human hepatoblastoma HepG2 cells and a model system, we showed that the activity of the hSR-BI promoter is subject to negative regulation by HNF- 4. These data are in agreement with previous studies which had shown that inactivation of the HNF-4 gene in the mouse is associated with a drastic increase in hepatic SR-BI levels. Silencing of the endogenous HNF-4 gene in HepG2 cells by shRNA increased the SR-BI mRNA levels whereas overexpression of HNF-4 repressed endogenous SR-BI gene expression. Promoter deletion analysis established that the region of the human SR-BI promoter between nucleotides - 201 and -62 is sufficient to mediate repression by HNF-4. By in vitro and in vivo protein-DNA interaction assays we demonstrated recruitment of HNF-4 to multiple sites on the hSR-BI promoter, suggesting a direct mechanism of repression of SR-BI gene transcription by HNF-4 in the liver. In summary, this is the first report showing that glucocorticoids suppress SR-BI gene expression in steroidogenic tissues suggesting that these tissues maintain steroid hormone homeostasis by prohibiting SR-BI-mediated highdensity lipoprotein cholesterol uptake when the endogenous levels of glucocorticoids are elevated. This is also the first report showing that HNF-4, a known transcriptional activator of apolipoprotein gene expression in the liver, acts as a direct negative regulator of SR-BI activity via a direct mechanism. 12 Understanding in depth the molecular mechanisms that regulate SR-BI gene expression in steroidogenic tissues and in the liver and the role of specific transcription factors might offer new therapeutic avenues for the prevention or treatment of atherosclerosis and might be useful in assessments of risk factors and drug efficacy.

Language

Greek

Subject

Atherosclerosis

Endocrine system.

Glucocorticoids

HNF-4

SR-BI

Αθηροσκλήρωση

Γλυκοκορτικοειδή

Ενδοκρινικό σύστημα

Issue date