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Znf202 affects high density lipoprotein cholesterol levels and promotes hepatosteatosis in hyperlipidemic mice.

Vrins CL, Out R, van Santbrink P, van der Zee A, Mahmoudi T, Groenendijk M, Havekes LM, van Berkel TJ, Willems van Dijk K, Biessen EA - PLoS ONE (2013)

Bottom Line: The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified.Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands. c_vrins@hotmail.com

ABSTRACT

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.

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Related in: MedlinePlus

Znf202 overexression results in the reduced lipid related gene expression and affects plasma lipid levels in vivo.mRNA levels relative to control genes in livers from Ldlr- 24 hrs after injection with Ad.Znf202 (filled bars) or Ad.mock (empty bars) (A). Plasma samples were taken before (day 0) and 1 day after injection with Ad.Znf202-treated (filled bars) and Ad.mock-treated animals (open bars) to determine the total plasma cholesterol and triglycerides content. Plasma samples obtained from day 1 after injection with Ad.Znf202 (triangles) or with Ad.mock (squares) were also analyzed for lipoprotein profile (B). Data are means ± S.D (n = 5). * and ** indicates p<0.05 and p<0.001 respectively.
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pone-0057492-g005: Znf202 overexression results in the reduced lipid related gene expression and affects plasma lipid levels in vivo.mRNA levels relative to control genes in livers from Ldlr- 24 hrs after injection with Ad.Znf202 (filled bars) or Ad.mock (empty bars) (A). Plasma samples were taken before (day 0) and 1 day after injection with Ad.Znf202-treated (filled bars) and Ad.mock-treated animals (open bars) to determine the total plasma cholesterol and triglycerides content. Plasma samples obtained from day 1 after injection with Ad.Znf202 (triangles) or with Ad.mock (squares) were also analyzed for lipoprotein profile (B). Data are means ± S.D (n = 5). * and ** indicates p<0.05 and p<0.001 respectively.

Mentions: Despite hypoalphalipoproteinemia and the dramatic changes in lipid levels, especially in Ldlr−/−, we did not observe any of the expected, repressive effects of Znf202 on hepatic expression of target genes in vivo at 5 day post-transduction. In addition, several lipid related nuclear receptors were dysregulated in Znf202 overexpressing Ldlr−/− mice, although none had been reported to be Znf202 responsive. Conceivably, the initial and suppressive effects of Znf202 overexpression are overruled by secondary effects, which may underly the severe phenotype observed in Ldlr−/− mice. To address this notion, we have monitored hepatic gene expression patterns and lipid homeostasis in the mouse model that was mostly affected, the Ldlr−/−, at 24 hours after transduction. In line with the in vitro results, this increase in hepatic Znf202 gene expression (Fig. S1; from ΔCT = −14 to ΔCT = −11) resulted in the reduced expression of several genes within both apolipoprotein gene clusters in the liver (Fig 5A). The mRNA levels of Apoa1, Apoc1, Apoc1, and Apoe were approximately 2-fold reduced. Interestingly, we also observed a lowered expression (>70%) of the sterol transporter genes Abcg5 and Abcg8 and the bile salt efflux protein (Bsep). Most notably, the expression of the bile acid synthesis gene cholesterol 7α-hydroxylase (Cyp7a1) was more that 1000 fold reduced upon treatment with Ad.Znf202 (P<0.001). The attenuated expression of these bile flux genes suggests that hepatic overexpression of Znf202 affects the biliary secretion. The expression of several lipid related transcription factors such as Fxr, Pparα, and Srepb1 was also reduced by hepatic Znf202 overexpression (table S4). However, a well described target gene of Znf202, Abca1, was unaffected. Additionally, the expression of HMG-CoA reductase, an important enzyme in cholesterol synthesis, was increased.


Znf202 affects high density lipoprotein cholesterol levels and promotes hepatosteatosis in hyperlipidemic mice.

Vrins CL, Out R, van Santbrink P, van der Zee A, Mahmoudi T, Groenendijk M, Havekes LM, van Berkel TJ, Willems van Dijk K, Biessen EA - PLoS ONE (2013)

Znf202 overexression results in the reduced lipid related gene expression and affects plasma lipid levels in vivo.mRNA levels relative to control genes in livers from Ldlr- 24 hrs after injection with Ad.Znf202 (filled bars) or Ad.mock (empty bars) (A). Plasma samples were taken before (day 0) and 1 day after injection with Ad.Znf202-treated (filled bars) and Ad.mock-treated animals (open bars) to determine the total plasma cholesterol and triglycerides content. Plasma samples obtained from day 1 after injection with Ad.Znf202 (triangles) or with Ad.mock (squares) were also analyzed for lipoprotein profile (B). Data are means ± S.D (n = 5). * and ** indicates p<0.05 and p<0.001 respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585336&req=5

pone-0057492-g005: Znf202 overexression results in the reduced lipid related gene expression and affects plasma lipid levels in vivo.mRNA levels relative to control genes in livers from Ldlr- 24 hrs after injection with Ad.Znf202 (filled bars) or Ad.mock (empty bars) (A). Plasma samples were taken before (day 0) and 1 day after injection with Ad.Znf202-treated (filled bars) and Ad.mock-treated animals (open bars) to determine the total plasma cholesterol and triglycerides content. Plasma samples obtained from day 1 after injection with Ad.Znf202 (triangles) or with Ad.mock (squares) were also analyzed for lipoprotein profile (B). Data are means ± S.D (n = 5). * and ** indicates p<0.05 and p<0.001 respectively.
Mentions: Despite hypoalphalipoproteinemia and the dramatic changes in lipid levels, especially in Ldlr−/−, we did not observe any of the expected, repressive effects of Znf202 on hepatic expression of target genes in vivo at 5 day post-transduction. In addition, several lipid related nuclear receptors were dysregulated in Znf202 overexpressing Ldlr−/− mice, although none had been reported to be Znf202 responsive. Conceivably, the initial and suppressive effects of Znf202 overexpression are overruled by secondary effects, which may underly the severe phenotype observed in Ldlr−/− mice. To address this notion, we have monitored hepatic gene expression patterns and lipid homeostasis in the mouse model that was mostly affected, the Ldlr−/−, at 24 hours after transduction. In line with the in vitro results, this increase in hepatic Znf202 gene expression (Fig. S1; from ΔCT = −14 to ΔCT = −11) resulted in the reduced expression of several genes within both apolipoprotein gene clusters in the liver (Fig 5A). The mRNA levels of Apoa1, Apoc1, Apoc1, and Apoe were approximately 2-fold reduced. Interestingly, we also observed a lowered expression (>70%) of the sterol transporter genes Abcg5 and Abcg8 and the bile salt efflux protein (Bsep). Most notably, the expression of the bile acid synthesis gene cholesterol 7α-hydroxylase (Cyp7a1) was more that 1000 fold reduced upon treatment with Ad.Znf202 (P<0.001). The attenuated expression of these bile flux genes suggests that hepatic overexpression of Znf202 affects the biliary secretion. The expression of several lipid related transcription factors such as Fxr, Pparα, and Srepb1 was also reduced by hepatic Znf202 overexpression (table S4). However, a well described target gene of Znf202, Abca1, was unaffected. Additionally, the expression of HMG-CoA reductase, an important enzyme in cholesterol synthesis, was increased.

Bottom Line: The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified.Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands. c_vrins@hotmail.com

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus