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Identifier 000347028
Title Μονοπάτια σηματοδότησης που ρυθμίζουν την έκφραση των γονιδίων των απολιποπρωτεϊνών του ανθρώπου
Author Νικολαϊδου-Νεοκοσμίδου, Βαρβάρα
Thesis advisor Καρδάσης, Δημήτριος
Reviewer Ζαννής, Βασίλειος
Ταλιανίδης, Γ.
Αλεξανδράκης, Δ.
Γραβάνης, Αχιλλέας
Ηλιόπουλος, Αριστείδης
Στουρνάρας, Χρήστος
Τσατσάνης, Χρήστος
Abstract The focus of the present work is the elucidation of the role of signal transduction pathways involving pro-inflammatory cytokines and hormone nuclear receptors in the transcriptional regulation of the genes coding for apolipoproteins in human hepatocytes. Apolipoproteins, along with plasma enzymes, lipid transfer proteins and the lipoprotein receptors participate in the biogenesis and catabolism of lipoproteins. In principle, changes in the regulation of synthesis of any apolipoprotein or another protein of the lipoprotein system may affect the concentration or the function of a specific group of lipoproteins, and may in some instances contribute to the pathogenesis of hyperilipidemia or even atherosclerosis. In the first part, we studied the effect of the pro-inflammatory cytokine TNFα (tumour necrosis factor α) on the regulation of apoCIII gene expression in human hepatocytes. We found that there is an antagonistic effect between TNFα and the antiinflammatory cytokine TGFβ (Transforming Growth Factor β) for the regulation of apoCIII gene expression. We showed that TNFα was a strong inhibitor of the activity of apolipoprotein promoters that harbour HNF-4α (hepatocyte nuclear factor 4) binding sites and this inhibition required HNF-4α, which is a key regulator of liverspecific gene expression in mammals. Using specific inhibitors of TNFα-induced signalling pathways, it was shown that inhibition of the apoCIII promoter by TNFα involved NF-κB (nuclear factor κB). By using LMP1 (Latent membrane protein 1) of the Epstein–Barr virus, which is an established potent activator of NF-κB as well as wild-type forms of various NF-κB signalling mediators, we proved that NF-κB is responsible, at least in part, for the strong inhibition of both apoCIII promoter’s activity and HNF-4α’ s transactivation function. We found that TNFα had no effect on the stability or the nuclear localization of HNF-4α in HepG2 cells, but inhibited, at least in part, the binding properties of HNF-4α to the proximal apoCIII HRE (hormone response element). By using the yeast-transactivator-GAL4 system, we showed that both AF-1 and AF-2 (activation functions 1 and 2) of HNF-4α are inhibited by TNFα and that this inhibition was abolished by overexpression of different HNF-4α co-activators, including PGC-1α (peroxisome-proliferatoractivated- receptor-γ co-activator 1α), CBP [CREB (cAMP-response-element-binding protein) binding protein] and SRC3 (steroid receptor co-activator 3). By using in vitro experimental procedures such as GST-pull downs, we showed that the p65/Rel A subunit of NF-κB interacts efficiently with HNF-4α. In the second part, we studied the effect of TNFα on the regulation of gene expression of apolipoproteins, of hormone nuclear receptors and of their co-regulators in human hepatocytes. By using RT-PCR (Reverse Transcription-PCR) assays, we found that the expression patterns of some of these genes show an early and a late response to TNFα. This could be attributed to different post-translational modifications of regulatory proteins triggered by TNFα’s signaling pathways that could affect the expression of target genes in a positive or negative way. By examining the expression pattern of the nuclear receptor inhibitor SHP (Small Heterodimer Partner) and the co-activator PGC-1α, which are very critical nuclear receptor co-regulators and by using specific inhibitors of the MEK1/2 kinase, we showed that TNFα regulates SHP and PGC-1α gene expression through the MEK1/2 signalling pathway. In the third part, we investigated more thoroughly the effect of TNFα on the regulation of SHP gene expression. We found that TNFα regulates SHP gene expression not only by the MEK1/2 pathway, but also by the activation of NF-κB and that both of these pathways have an inhibitory effect on SHP promoter activity. We also determined a region of the SHP promoter, between nucleotides -1383/-865, that mediates the inhibitory effect of NF-κB. We also showed that HNF-4α and its coactivator PGC-1α are strong activators of SHP gene expression in hepatocytes. In the fourth part, we focused on the role of Tpl2/Cot, a protein that belongs to the MAP3K family and is activated by pro-inflammatory cytokines such as TNFα, in apolipoprotein gene regulation and nuclear receptor activity in hepatic cells. We showed that Tpl2 strongly inhibited the constitutive activity of the HNF-4α and the HNF-4α-mediated transactivation of the human apoCIII promoter in human hepatoma HepG2 cells. Using specific inhibitors and dominant negative mutants we showed that the NF-κB and the MEK1/ERK pathways are not required for the inhibition of HNF- 4α activity by Tpl2. We showed that the inhibition of HNF-4α activity by Tpl2/Cot is not due to HNF-4α’s decreased DNA binding efficiency but because of the inability of HNF-4α to interact with its co-activator PGC-1α. The expression of a dominant negative mutant of Tpl2 or a short-hairpin mRNA of COT strongly activated all apolipoprotein genes tested in HepG2 cells confirming the important role of endogenous COT in apoC-III and possibly in other apolipoprotein gene regulation in hepatocytes. Finally, we showed that Tpl2/Cot inhibited the inducible activity of the RXRα and of the T3Rβ hormone nuclear receptors in GAL4-based transactivation assays as well as the RXRα/T3Rβ-mediated induction of apoCII gene expression suggesting that Tpl2/Cot has a broader role in hormone nuclear receptor activity in hepatic cells. In the fifth part, we studied the role of phosphorylation in nuclear hormone receptor activity. We found that treatment of HepG2 cells with the chemotherapeutic agent 5-Fluorouracil caused the proline-directed Ser/Thr phosphorylation of HNF-4α and inhibited its activity as well as the activity of the apoCIII promoter. We also investigated the possible role of prolyl cis/trans isomerase Pin1 in the transcriptional activity of the hormone nuclear receptors HNF-4α and RXRα. In both cases we found that the presence of Pin1 had an inhibitory effect on the transactivation efficiency of phospshorylated (pSer/Thr-Pro) nuclear receptors. Finally, we studied the involvement of Pin1 in the transcriptional regulation of various apolipoprotein promoters and we found that Pin1 seems to control, in a positive way, the expression of the apoE gene, an observation that needs to be investigated further.
Physical description 227 σ. : πιν. ; 30 εκ.
Language Greek
Subject Lipoproteins
Issue date 2007-12-14
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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