1st chapter: Role of the transcriptional factor c-Jun in the regulation of genes that affect
lipid homeostasis in vivo
c-Jun (cellular Jun) is a transcription factor activated by phosphorylation by the stress
activated protein kinase/c-Jun-N-terminal-kinase pathway in response to extracellular signals and
cytokines. We show that adenovirus mediated gene transfer of dn-c-Jun (dominant negative form
of c-Jun) in C57BL/6 mice increased greatly apoE (apolipoprotein E) hepatic mRNA and plasma
levels, increased plasma cholesterol, triglyceride and VLDL (very low density lipoprotein) levels
and resulted in the accumulation of discoidal HDL (high density lipoprotein). A similar but more
severe phenotype was generated by overexpression of the mouse apoE in C57BL/6 mice,
suggesting that dyslipidemia induced by dn-c-Jun was the result of apoE overexpression.
Unexpectedly, infection of apoE-/- mice with adenovirus expressing dn-c-Jun reduced by
70% plasma cholesterol, suggesting that dn-c-Jun affected other genes that control plasma
cholesterol levels. To identify these genes we performed whole genome expression analysis
(34,000 genes) of isolated livers from two groups of five apoE-/- mice, infected with adenoviruses
expressing either the dn-c-Jun or the green fluorescence protein. Bioinformatical analysis and
Northern blotting validation revealed that dn-c-Jun increased 40-fold the apoE mRNA and
reduced by 70% the Scd-1 (stearoyl-CoA-desaturase 1) mRNA. The involvement of Scd-1 in
lowering plasma cholesterol was confirmed by restoration of high cholesterol levels of apoE-/-
mice following coinfection with adenoviruses expressing dn-c-Jun and Scd-1.
In conclusion, dn-c-Jun appears to trigger two opposing events in mice that affect plasma
cholesterol and triglyceride levels: One results in apoE overexpression and triggers dyslipidemia
and the other results in inhibition of Scd-1 and offsets dyslipidemia.
The ability of the dn-c-Jun to upregulate the apoE gene expression is unique for the dn-c-
Jun molecule since the wild type c-Jun as well as a constitutively active and an inactive mutant of
the c-Jun did not affect the hepatic apoE mRNA levels.
2nd chapter: Regulation of the expression of apoE and other apoliopoprotein genes in
HepG2 cells by proteins of the JNK/SAPK signaling pathway and the nuclear receptor
RxRα
In vitro experiments were performed in HepG2 cells to assess the role of proteins of the
JNK/SAPK signaling pathway in the regulation of apoE and other apolipoprotein genes.
Infection of HepG2 cells with recombinant adenoviruses expressing a dominant negative form of
the JNK2 (dn-JNK) or a constitutively active form of this protein (JNK2α2) showed that the dn-
JNK reduced the apoE gene expression whereas JNK2α2 increased the apoE gene expression.
Admininstration of a recombinant adenovirus expressing the Peptidyl-prolyl isomerase Pin1,
which contributes to the activation of the phosphorylated c-Jun, in HepG2 cells that were alos
treated with 5 Fluoruracil (5-FU) increased the apoE mRNA levels. Similar analyses for apoA-I
and apoC-III showed that the dn-JNK did not change significantly the expression of these genes.
However Pin1 in combination with 5-FU reduced significantly both apoA-I and apoC-III gene
expression.
In other experiments HepG2 cells were infected with recombinant adenoviruses
expressing the dn-c-Jun and a dominant negative mutant of the Sp1 (dn-Sp1). It was found that
the upregulation of the apoE gene expression by the dn-c-Jun was not affecred by the dn-Sp1
suggesting that the dn-c-Jun acts independently of the Sp1.
Finally, infection of HepG2 cells with recombinant adenoviruses expressing either the
wild type RxRα or a dominant negative mutant form of the RxRα (dn-RxRα) showed that the wild
type RxRα in combination with the RxRα ligand, 9-cis-retinoic acid, seems to increase the
expression of the apoE, apoA-I and apoC-III genes.
Further experiments are required to assess the role of these factors in apoE gene
regulation.
3rd chapter: Aminoacids Leu261, Trp264 and Phe265 of the human apoE account for the
induction of dyslipidemia and affect the formation of the apoE-containing HDL
Overexpression of apolipoprotein E (apoE) induces hypertriglyceridemia in apoE-deficient
mice, which is abrogated by deletion of the carboxy-terminal segment 260-299.
We have used adenovirus-mediated gene transfer in apoE-/- and apoA-I-/- mice to test the
effect of three sets of apoE mutations within the 261-265 region on the induction of
hypertriglyceridemia, the esterification of cholesterol of very low density lipoprotein (VLDL) and
high density lipoprotein (HDL) and the formation of spherical or discoidal apoE-containing HDL.
A single (apoE4 [Phe265Ala]) amino acid substitution induced hypertriglyceridemia in
apoE-/- or apoA-I-/- mice, promoted the accumulation of free cholesterol in the very low density
lipoprotein (VLDL) and HDL region and decreased HDL cholesterol levels. A double (apoE4
[Leu261Ala, Trp264Ala]) substitution induced milder hypertriglyceridemia and increased HDL
cholesterol levels. A triple substitution (apoE4 [Leu261Ala, Trp264Ala, Phe265Ala] or apoE2
[Leu261Ala, Trp264Ala, Phe265Ala]) did not induce hypertriglyceridemia and increased greatly
the HDL cholesterol levels. Electron microscopy (EM) analysis of the HDL fractions showed that
apoE4 [Leu261Ala, Trp264Ala, Phe265Ala] and the apoE2 [Leu261Ala, Trp264Ala, Phe265Ala]
contained spherical HDL, the apoE4 [Leu261Ala, Trp264Ala] contained mostly spherical and few
discoidal HDL particles and apoE4 [Phe265Ala] contained discoidal HDL. A quadruplex (apoE4
[Leu261Ala, Trp264Ala, Phe265Ala, Leu268Ala]) substitution reduced cholesterol and induced
mild hypertriglyceridemia.
A double substitution in the first helix of the N-terminal region of the apoE4 (apoE4
[Arg25Ala, Arg32Ala]) did not reduce the plasma cholesterol levels and increased triglycerides.
We conclude that residues Leu261, Trp264 and Phe265 account for the apoE-induced
hypertriglyceridemia, the accumulation of free cholesterol in VLDL and HDL and the formation of
discoidal HDL. Substitution of these residues by Ala improves the apoE functions by preventing
hypertriglyceridemia and promoting formation of spherical apoE-containing HDL. This beneficial
phenotype is regardless of the apoE phenotype.
(EN)
