Abstract
c-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 the dominant negative form of c-Jun (dn-c-Jun) in C57BL/6 mice increased greatly apoE hepatic mRNA and plasma levels, increased plasma cholesterol, triglyceride, and very low density lipoprotein levels, and resulted in the accumulation of discoidal high density lipoprotein particles. 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 plasma cholesterol by 70%, 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. Bioinformatic 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 as follows: one results in apoE overexpression and triggers dyslipidemia and the other results in inhibition of Scd-1 and offsets dyslipidemia.
Highlights
Jun N-terminal kinase (JNK) and c-Jun as well as signals that activate them have been linked to the regulation of genes involved in lipid and lipoprotein homeostasis and in atherosclerosis (20 –31)
Form of c-Jun in C57BL/6 Mice Increases the Hepatic mRNA 2F), whereas the equivalent fractions 5– 8 that were obtained and Plasma ApoE Levels—We used adenovirus-mediated gene from plasma of C57BL/6 mice infected with control adenovirus transfer of dn-c-Jun in C57BL/6 mice to assess its effects on expressing the GFP formed spherical HDL particles (Fig. 2E)
A similar up-regulation of apolipoprotein E (apoE) mRNA infected with adenovirus expressing dn-c-Jun as compared levels was observed in HepG2 cells infected with the Ad-dn- with mice infected with the control adenovirus expressing GFP
Summary
Materials—Reagents were purchased from the following commercial sources. Restriction enzymes and modifying enzymes (T4 DNA ligase, Klenow fragment of DNA polymerase I) were purchased from New England Biolabs. The medium was changed to 2% heat-inactivated horse serum, and the cells were infected in at least duplicates with control adenovirus that expresses the green fluorescent protein (Ad-GFP) and the adenoviruses expressing the dn-c-Jun form (Ad-dn-c-Jun) and the wild-type Scd-1 at a multiplicity of infection of 5. A two-class unpaired data analysis was performed on Assessment—Total RNA was extracted from the livers of 10 normalized and filtered data, using a ⌬ threshold of 1.571 Five of these mice were infected with Ad-dn-c- “⌬” parameter enables the user to examine the effect of the. Analysis of the cytoplasmic and nuclear extracts of infected HepG2 cells by SDS-PAGE and Western blotting showed that the dn-c-Jun was expressed efficiently and was found both in cytoplasm and the nucleus (supplemental Fig. 2)
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