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Regulation of IGFBP-2 expression during fasting.

Kang HS, Kim MY, Kim SJ, Lee JH, Kim YD, Seo YK, Bae JH, Oh GT, Song DK, Ahn YH, Im SS - Biochem. J. (2015)

Bottom Line: Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes.To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation.These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

View Article: PubMed Central - PubMed

Affiliation: *Department of Physiology, Keimyung University School of Medicine, Daegu 704-701, Republic of Korea.

ABSTRACT
Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2), one of the most abundant circulating IGFBPs, is known to attenuate the biological action of IGF-1. Although the effect of IGFBP-2 in preventing metabolic disorders is well known, its regulatory mechanism remains unclear. In the present study, we demonstrated the transcriptional regulation of the Igfbp-2 gene by peroxisome-proliferator-activated receptor (PPAR) α in the liver. During fasting, both Igfbp-2 and PPARα expression levels were increased. Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes. However, Igfbp-2 gene expression in Pparα mice was not affected by fasting or Wy14643. In addition, through transient transfection and chromatin immunoprecipitation assay in fasted livers, we determined that PPARα bound to the putative PPAR-responsive element between -511 bp and -499 bp on the Igfbp-2 gene promoter, indicating that the Igfbp-2 gene transcription is activated directly by PPARα. To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation. No inhibition was observed in the hepatocytes isolated from Pparα mice. These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

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Identification of PPRE in the mIgfbp-2 promoter(A) Comparison of putative PPRE with consensus PPRE sequences. A proposed PPARα-binding element is shaded on the Igfbp-2 promoter between −511 and −499. The numbers indicate the distance in nucleotides from the transcription start site (+1) of the mouse Igfbp-2 gene. (B) Effects of PPARα on promoter reporter activities in deletion constructs of the Igfbp-2 gene. Deletion construct of the mIgfbp-2 promoter was transiently co-transfected with pcDNA3 (open bars) or PPARα expression vector (closed bars) in HEK-293T cells. After 24 h, media were changed to contain 20 μM Wy14643. Luciferase activity was normalized to β-galactosidase activity to correct for transfection efficiency. (C) Internal deletion constructs for the Igfbp-2 promoter were analysed for promoter activity. (D and E) ChIP assay. Mice were fasted for 24 h and refed for 12 h. Chromatins were isolated from mice livers and ChIP assay was performed. Input represents 10% of purified DNA in each sample. Nuclear extracts from mice livers were immunoprecipitated with anti-PPARα antibody and purified DNA samples were used to perform qPCR with primers binding to the putative PPRE regions on the mIgfbp-2 (D) and mG6pc (E) gene promoters. All data are representative of at least three independent experiments. *P<0.05 compared with untreated control.
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Figure 3: Identification of PPRE in the mIgfbp-2 promoter(A) Comparison of putative PPRE with consensus PPRE sequences. A proposed PPARα-binding element is shaded on the Igfbp-2 promoter between −511 and −499. The numbers indicate the distance in nucleotides from the transcription start site (+1) of the mouse Igfbp-2 gene. (B) Effects of PPARα on promoter reporter activities in deletion constructs of the Igfbp-2 gene. Deletion construct of the mIgfbp-2 promoter was transiently co-transfected with pcDNA3 (open bars) or PPARα expression vector (closed bars) in HEK-293T cells. After 24 h, media were changed to contain 20 μM Wy14643. Luciferase activity was normalized to β-galactosidase activity to correct for transfection efficiency. (C) Internal deletion constructs for the Igfbp-2 promoter were analysed for promoter activity. (D and E) ChIP assay. Mice were fasted for 24 h and refed for 12 h. Chromatins were isolated from mice livers and ChIP assay was performed. Input represents 10% of purified DNA in each sample. Nuclear extracts from mice livers were immunoprecipitated with anti-PPARα antibody and purified DNA samples were used to perform qPCR with primers binding to the putative PPRE regions on the mIgfbp-2 (D) and mG6pc (E) gene promoters. All data are representative of at least three independent experiments. *P<0.05 compared with untreated control.

Mentions: To identify the fundamental molecular mechanism through which PPARα regulates Igfbp-2 gene transcription, the mouse Igfbp-2 (mIgfbp-2) gene promoter was transiently transfected into human embryonic kidney (HEK)-293T-cells. As shown in Figure 3(A), computer analysis with consensus PPRE sequence showed a highly conserved putative PPRE on the Igfbp-2 promoter. A proposed PPARα-binding element is shaded in the Igfbp-2 promoter between −511 bp and −499 bp. The transcriptional activity of mIgfbp-2 in response to Wy14643 treatment was highest with a full-length gene promoter and was diminished markedly with deletion up to −444 bp (Figure 3B). Moreover, internal deletion of the putative PPRE between −511 bp and −499 bp from the full-length mIgfbp-2 resulted in decreased promoter activity (Figure 3C). These data collectively suggest that the putative PPRE is located between the −511 and −499 bp regions of the mIgfbp-2 gene promoter. We next performed ChIP assays in mouse livers to verify PPARα binding to the mIgfbp-2 gene promoter at the chromatin level. PPARα occupancy was greater on the mIgfbp-2 promoter (Figure 3D). A known PPRE region on the G6pc gene promoter was used as a positive control in the ChIP assay (Figure 3E). These results demonstrate that Igfbp-2 gene expression is regulated through the direct binding of PPARα to the Igfbp-2 promoter.


Regulation of IGFBP-2 expression during fasting.

Kang HS, Kim MY, Kim SJ, Lee JH, Kim YD, Seo YK, Bae JH, Oh GT, Song DK, Ahn YH, Im SS - Biochem. J. (2015)

Identification of PPRE in the mIgfbp-2 promoter(A) Comparison of putative PPRE with consensus PPRE sequences. A proposed PPARα-binding element is shaded on the Igfbp-2 promoter between −511 and −499. The numbers indicate the distance in nucleotides from the transcription start site (+1) of the mouse Igfbp-2 gene. (B) Effects of PPARα on promoter reporter activities in deletion constructs of the Igfbp-2 gene. Deletion construct of the mIgfbp-2 promoter was transiently co-transfected with pcDNA3 (open bars) or PPARα expression vector (closed bars) in HEK-293T cells. After 24 h, media were changed to contain 20 μM Wy14643. Luciferase activity was normalized to β-galactosidase activity to correct for transfection efficiency. (C) Internal deletion constructs for the Igfbp-2 promoter were analysed for promoter activity. (D and E) ChIP assay. Mice were fasted for 24 h and refed for 12 h. Chromatins were isolated from mice livers and ChIP assay was performed. Input represents 10% of purified DNA in each sample. Nuclear extracts from mice livers were immunoprecipitated with anti-PPARα antibody and purified DNA samples were used to perform qPCR with primers binding to the putative PPRE regions on the mIgfbp-2 (D) and mG6pc (E) gene promoters. All data are representative of at least three independent experiments. *P<0.05 compared with untreated control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403943&req=5

Figure 3: Identification of PPRE in the mIgfbp-2 promoter(A) Comparison of putative PPRE with consensus PPRE sequences. A proposed PPARα-binding element is shaded on the Igfbp-2 promoter between −511 and −499. The numbers indicate the distance in nucleotides from the transcription start site (+1) of the mouse Igfbp-2 gene. (B) Effects of PPARα on promoter reporter activities in deletion constructs of the Igfbp-2 gene. Deletion construct of the mIgfbp-2 promoter was transiently co-transfected with pcDNA3 (open bars) or PPARα expression vector (closed bars) in HEK-293T cells. After 24 h, media were changed to contain 20 μM Wy14643. Luciferase activity was normalized to β-galactosidase activity to correct for transfection efficiency. (C) Internal deletion constructs for the Igfbp-2 promoter were analysed for promoter activity. (D and E) ChIP assay. Mice were fasted for 24 h and refed for 12 h. Chromatins were isolated from mice livers and ChIP assay was performed. Input represents 10% of purified DNA in each sample. Nuclear extracts from mice livers were immunoprecipitated with anti-PPARα antibody and purified DNA samples were used to perform qPCR with primers binding to the putative PPRE regions on the mIgfbp-2 (D) and mG6pc (E) gene promoters. All data are representative of at least three independent experiments. *P<0.05 compared with untreated control.
Mentions: To identify the fundamental molecular mechanism through which PPARα regulates Igfbp-2 gene transcription, the mouse Igfbp-2 (mIgfbp-2) gene promoter was transiently transfected into human embryonic kidney (HEK)-293T-cells. As shown in Figure 3(A), computer analysis with consensus PPRE sequence showed a highly conserved putative PPRE on the Igfbp-2 promoter. A proposed PPARα-binding element is shaded in the Igfbp-2 promoter between −511 bp and −499 bp. The transcriptional activity of mIgfbp-2 in response to Wy14643 treatment was highest with a full-length gene promoter and was diminished markedly with deletion up to −444 bp (Figure 3B). Moreover, internal deletion of the putative PPRE between −511 bp and −499 bp from the full-length mIgfbp-2 resulted in decreased promoter activity (Figure 3C). These data collectively suggest that the putative PPRE is located between the −511 and −499 bp regions of the mIgfbp-2 gene promoter. We next performed ChIP assays in mouse livers to verify PPARα binding to the mIgfbp-2 gene promoter at the chromatin level. PPARα occupancy was greater on the mIgfbp-2 promoter (Figure 3D). A known PPRE region on the G6pc gene promoter was used as a positive control in the ChIP assay (Figure 3E). These results demonstrate that Igfbp-2 gene expression is regulated through the direct binding of PPARα to the Igfbp-2 promoter.

Bottom Line: Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes.To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation.These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

View Article: PubMed Central - PubMed

Affiliation: *Department of Physiology, Keimyung University School of Medicine, Daegu 704-701, Republic of Korea.

ABSTRACT
Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2), one of the most abundant circulating IGFBPs, is known to attenuate the biological action of IGF-1. Although the effect of IGFBP-2 in preventing metabolic disorders is well known, its regulatory mechanism remains unclear. In the present study, we demonstrated the transcriptional regulation of the Igfbp-2 gene by peroxisome-proliferator-activated receptor (PPAR) α in the liver. During fasting, both Igfbp-2 and PPARα expression levels were increased. Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes. However, Igfbp-2 gene expression in Pparα mice was not affected by fasting or Wy14643. In addition, through transient transfection and chromatin immunoprecipitation assay in fasted livers, we determined that PPARα bound to the putative PPAR-responsive element between -511 bp and -499 bp on the Igfbp-2 gene promoter, indicating that the Igfbp-2 gene transcription is activated directly by PPARα. To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation. No inhibition was observed in the hepatocytes isolated from Pparα mice. These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

Show MeSH
Related in: MedlinePlus