Znf202 Affects High Density Lipoprotein Cholesterol Levels and Promotes Hepatosteatosis in Hyperlipidemic Mice

Background: The zinc finger protein Znf202 is a transcriptional suppressor of lipid related genes and has been linked to hypoalphalipoproteinemia. A functional role of Znf202 in lipid metabolism in vivo still remains to be established. Methodology and Principal Findings: We generated mouse Znf202 expression vectors, the functionality of which was established in several in vitro systems. Next, effects of adenoviral znf202 overexpression in vivo were determined in normo- as well as hyperlipidemic mouse models. Znf202 overexpression in mouse hepatoma cells mhAT3F2 resulted in downregulation of members of the Apoe/c1/c2 and Apoa1/c3/a4 gene cluster. The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified. Adenoviral Znf202 transfer to Ldlr-/- mice resulted in downregulation of apoe, apoc1, apoa1, and apoc3 within 24 h after gene transfer. Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated. At 5 days post-infection, the expression of the aforementioned genes was normalized, but mice had developed severe hepatosteatosis accompanied by hypercholesterolemia and hypoalphalipoproteinemia. A much milder phenotype was observed in wildtype mice after 5 days of hepatic Znf202 overexpression. Interestingly and similar to Ldl-/- mice, HDL-cholesterol levels in wildtype mice were lowered after hepatic Znf202 overexpression. Conclusion/Significance: Znf202 overexpression in vivo reveals an important role of this transcriptional regulator in liver lipid homeostasis, while firmly establishing the proposed key role in the control of HDL levels

Hepatic Znf202 overexpression causes hepatosteatosis in <i>Ldlr−/−</i> mice only.

<p>Livers were isolated from <i>Ldlr−/−</i> and WT mice 5 days after injection with 2.10⁹pfu of Ad.Znf202 (filled bars) or Ad-mock (empty bars). Cryosections were prepared from Ldlr−/− liver samples and stained with Oil Red-O (A). Hepatic lipids were extracted from homogenized liver samples and cholesterol and TG concentrations were determined (B). Values are expressed as µg lipid per mg tissue protein and are means ± SD (n = 4). * indicates p<0.05.</p

The Znf202 specifically binds to the alleged response elements

<p>−<b>678 and</b> −<b>564 within the</b> −<b>705/−362 region of the mouse apoE promoter.</b> (A) DNA fragments used in this study containing the putative Znf202 binding sequence. The putative consensus sequences are underlined. (B) EMSA with labeled DNA fragments GnT (lanes 1–10), −678 (lanes 11–20), −564 (lanes 21–30), and PHO (lanes 31–35) described above. Reactions contained the indicated amount (0.1–0.6 µg) of whole cell extract made from either control 911 cells, or from 911 cells overexpressing Znf202. Competition experiments using 50-fold excess of unlabeled GnT oligonucleotide (lanes 4,7 and 10), −678 oligonucleotide (lanes 14,17 and 20), or -564 oligonucleotide (lanes 24,27 and 30) confirmed the specificity of Znf202 binding to both GnT elements. Arrowheads indicate the mobility of unbound DNA and Znf202 protein bound DNA.</p

Hepatic Znf202 overexpression reduces HDL-cholesterol in <i>Ldlr−/−</i> and WT mice.

<p>Blood samples were drawn from <i>Ldlr−/−</i> (n = 4; left panels) and WT mice (n = 4; right panels) 5 days after injection with 2.10⁹pfu of Ad.Znf202 (filled bars) or with Ad-mock (open bars) and derived plasma was analyzed for triglyceride and total cholesterol content (A). Lipoprotein profiles were determined from <i>Ldlr−/−</i> (left panels) and WT mice (right panels) 5 days after injection with Ad.Znf202 (triangles) or with Ad-Mock (squares). The elution fractions were tested for triglyceride and total cholesterol content (B). * and ** indicates p<0.05 and p<0.001, respectively.</p

Relative gene expression in livers 5 days after infection with Ad-mock or Ad-Znf202 in Ldlr−/− and wild type mice.

<p>Values are expressed as means ± SD.</p>*<p>Indicates a significant difference (p<0.05) between Ad.Znf202 treated animals and their corresponding Ad.mock treated controls.</p

Proposed mechanism for Znf202.

<p>Elevated hepatic levels of Znf202 downregulate bile flux genes. Together with an increase in cholesterol synthesis and attenuated bile acid synthesis, this could result in the observed lipid accumulation in the liver and increased VLDL secretion (A). Under normolipidemic conditions, feedback mechanisms are able to reverse most of the initial effects of Znf202 overexpression (B). These initial effects cannot be sufficiently restored in mice under the hyperlipidemic conditions caused by the low density lipoprotein receptor deficiency. As a result, the mice become more hyperlipidemic and the lipid accumulation in the liver is followed by hepatic steatosis (C).</p

Znf202 overexpression leads to repression of members of the <i>apoe/c1/c2</i> and <i>apoa1/c3/a4/a5</i> gene clusters in mhAT3F2 and inhibits mouse apoE promoter activity.

<p>(A) MhAT3F2 cells were transduced with Ad.Znf202 (black bars) or Ad.LacZ (grey bars)(MOI = 100) and mRNA levels were measured via quantitative real time PCR at 24 hours post-infection. Data represent average of four transductions for each group (mean ± S.D) and expressions are relative to HPRT. (B) MhAT3F2 cells were transfected with mouse apoE promotor-reporter constructs, carrying a 729 bp fragment of the mouse apoE promoter lacking the downstream intron-1 (−705 to +24) or truncated variants thereof. Co-transfection with pCMV-LacZ served as a control for transfection efficiency. Luciferase activity was normalized for β-galactosidase activity (n = 4, mean ± S.D.). (C) MhAT3F2 cells were transiently cotransfected with the indicated reporter constructs and Znf202 expression vector (black bars). As control expression vector pShuttleCMV-empty was used (grey bars). After 24 hours cells were harvested and luciferase activities (n = 4, mean ± S.D.) measured and normalized for protein concentration.</p