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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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Znf202 overexpression leads to repression of members of the apoe/c1/c2 and apoa1/c3/a4/a5 gene clusters in mhAT3F2 and inhibits mouse apoE promoter activity.(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.
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pone-0057492-g001: Znf202 overexpression leads to repression of members of the apoe/c1/c2 and apoa1/c3/a4/a5 gene clusters in mhAT3F2 and inhibits mouse apoE promoter activity.(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.

Mentions: The functionality of the generated recombinant plasmids and adenovirus carrying containing the Znf202 gene construct was verified in vitro. As readout, we analyzed the effect of Znf202 overexpression on the Apoe/c1/c2 and Apoa1/c3/a4/a5 gene clusters in mhAT3F2 cells. Cyclophylin or 36B4 expression by transduced mhAT3F2 cells was not affected by Ad.Znf202 transduction (Fig.1A). However, Znf202 overexpression did result in a significant downregulation of apolipoprotein genes from both clusters. RNA levels were reduced up to 50% (ApoC2 and ApoC3) and reached significance for all genes (p<0.05) except Apoa5.


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 overexpression leads to repression of members of the apoe/c1/c2 and apoa1/c3/a4/a5 gene clusters in mhAT3F2 and inhibits mouse apoE promoter activity.(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.
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Related In: Results  -  Collection

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pone-0057492-g001: Znf202 overexpression leads to repression of members of the apoe/c1/c2 and apoa1/c3/a4/a5 gene clusters in mhAT3F2 and inhibits mouse apoE promoter activity.(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.
Mentions: The functionality of the generated recombinant plasmids and adenovirus carrying containing the Znf202 gene construct was verified in vitro. As readout, we analyzed the effect of Znf202 overexpression on the Apoe/c1/c2 and Apoa1/c3/a4/a5 gene clusters in mhAT3F2 cells. Cyclophylin or 36B4 expression by transduced mhAT3F2 cells was not affected by Ad.Znf202 transduction (Fig.1A). However, Znf202 overexpression did result in a significant downregulation of apolipoprotein genes from both clusters. RNA levels were reduced up to 50% (ApoC2 and ApoC3) and reached significance for all genes (p<0.05) except Apoa5.

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