| 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.
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