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Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T 3

View Article: PubMed Central - PubMed

ABSTRACT

The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp − 612 and − 156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TRβ1 to the − 612 and − 156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

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In vitro confirmation of the role of PPARα and USF-2 on the T3-dependent activation of the LDL receptor gene. Studies were performed in H4IIE cells grown in a thyroid hormone deficient medium. A. Promoter analysis experiment. Cells were co-transfected with the WT LDL receptor promoter construct, the TRβ1-pCMVS4 and RXRα-pRcRSV expression plasmids, the Renilla plasmid, and the indicated siRNAs using Fugene 6 transfection reagent. Lysing of the cells was performed 48 hours after transfections. Some cells were treated with T3 (1 μM) and 9-cis retinoic acid (RA; 1 μM) for 16 hours prior to lysing. Luciferase assays were carried out as described in Materials and methods. B. Analysis of mRNA levels. Cells were transfected with control siRNAs or with siRNAs for either PPARα or USF-2 as described above. Treatment with T3 and RA was carried out as described in A. Cells were used in the preparation of RNA 24 hours after transfection. RNA samples were analyzed using real-time PCR. In both panels, the data are presented as mean ± SEM for three samples per treatment condition. p Values for the samples transfected with the control siRNA and treated with T3/RA were obtained by comparing to the control siRNA samples no treated with the hormones. All the other p values were obtained by comparing to the control siRNA sample treated with T3/RA.
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f0030: In vitro confirmation of the role of PPARα and USF-2 on the T3-dependent activation of the LDL receptor gene. Studies were performed in H4IIE cells grown in a thyroid hormone deficient medium. A. Promoter analysis experiment. Cells were co-transfected with the WT LDL receptor promoter construct, the TRβ1-pCMVS4 and RXRα-pRcRSV expression plasmids, the Renilla plasmid, and the indicated siRNAs using Fugene 6 transfection reagent. Lysing of the cells was performed 48 hours after transfections. Some cells were treated with T3 (1 μM) and 9-cis retinoic acid (RA; 1 μM) for 16 hours prior to lysing. Luciferase assays were carried out as described in Materials and methods. B. Analysis of mRNA levels. Cells were transfected with control siRNAs or with siRNAs for either PPARα or USF-2 as described above. Treatment with T3 and RA was carried out as described in A. Cells were used in the preparation of RNA 24 hours after transfection. RNA samples were analyzed using real-time PCR. In both panels, the data are presented as mean ± SEM for three samples per treatment condition. p Values for the samples transfected with the control siRNA and treated with T3/RA were obtained by comparing to the control siRNA samples no treated with the hormones. All the other p values were obtained by comparing to the control siRNA sample treated with T3/RA.

Mentions: To confirm the involvement of PPARα and USF-2 in the T3-dependent transcriptional activation of the hepatic LDL receptor gene, knockdown studies using siRNAs against these two transcription factors were carried out in H4IIE cells. The LDL receptor promoter activity and receptor mRNA levels obtained in the presence of the siRNAs against PPARα and USF-2 were directly compared with the results obtained in the presence of a negative control siRNA. In the presence of T3/9-cis retinoic acid (RA), the receptor promoter activity was reduced to about 30% of control when siRNAs against PPARα and USF-2 where introduced (Fig. 6A). In agreement, LDL receptor mRNA levels were reduced to about 50% of control by the same siRNAs (Fig. 6B).


Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T 3
In vitro confirmation of the role of PPARα and USF-2 on the T3-dependent activation of the LDL receptor gene. Studies were performed in H4IIE cells grown in a thyroid hormone deficient medium. A. Promoter analysis experiment. Cells were co-transfected with the WT LDL receptor promoter construct, the TRβ1-pCMVS4 and RXRα-pRcRSV expression plasmids, the Renilla plasmid, and the indicated siRNAs using Fugene 6 transfection reagent. Lysing of the cells was performed 48 hours after transfections. Some cells were treated with T3 (1 μM) and 9-cis retinoic acid (RA; 1 μM) for 16 hours prior to lysing. Luciferase assays were carried out as described in Materials and methods. B. Analysis of mRNA levels. Cells were transfected with control siRNAs or with siRNAs for either PPARα or USF-2 as described above. Treatment with T3 and RA was carried out as described in A. Cells were used in the preparation of RNA 24 hours after transfection. RNA samples were analyzed using real-time PCR. In both panels, the data are presented as mean ± SEM for three samples per treatment condition. p Values for the samples transfected with the control siRNA and treated with T3/RA were obtained by comparing to the control siRNA samples no treated with the hormones. All the other p values were obtained by comparing to the control siRNA sample treated with T3/RA.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121208&req=5

f0030: In vitro confirmation of the role of PPARα and USF-2 on the T3-dependent activation of the LDL receptor gene. Studies were performed in H4IIE cells grown in a thyroid hormone deficient medium. A. Promoter analysis experiment. Cells were co-transfected with the WT LDL receptor promoter construct, the TRβ1-pCMVS4 and RXRα-pRcRSV expression plasmids, the Renilla plasmid, and the indicated siRNAs using Fugene 6 transfection reagent. Lysing of the cells was performed 48 hours after transfections. Some cells were treated with T3 (1 μM) and 9-cis retinoic acid (RA; 1 μM) for 16 hours prior to lysing. Luciferase assays were carried out as described in Materials and methods. B. Analysis of mRNA levels. Cells were transfected with control siRNAs or with siRNAs for either PPARα or USF-2 as described above. Treatment with T3 and RA was carried out as described in A. Cells were used in the preparation of RNA 24 hours after transfection. RNA samples were analyzed using real-time PCR. In both panels, the data are presented as mean ± SEM for three samples per treatment condition. p Values for the samples transfected with the control siRNA and treated with T3/RA were obtained by comparing to the control siRNA samples no treated with the hormones. All the other p values were obtained by comparing to the control siRNA sample treated with T3/RA.
Mentions: To confirm the involvement of PPARα and USF-2 in the T3-dependent transcriptional activation of the hepatic LDL receptor gene, knockdown studies using siRNAs against these two transcription factors were carried out in H4IIE cells. The LDL receptor promoter activity and receptor mRNA levels obtained in the presence of the siRNAs against PPARα and USF-2 were directly compared with the results obtained in the presence of a negative control siRNA. In the presence of T3/9-cis retinoic acid (RA), the receptor promoter activity was reduced to about 30% of control when siRNAs against PPARα and USF-2 where introduced (Fig. 6A). In agreement, LDL receptor mRNA levels were reduced to about 50% of control by the same siRNAs (Fig. 6B).

View Article: PubMed Central - PubMed

ABSTRACT

The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp − 612 and − 156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TRβ1 to the − 612 and − 156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

No MeSH data available.


Related in: MedlinePlus