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AMP-activated protein kinase-regulated activation of the PGC-1alpha promoter in skeletal muscle cells.

Irrcher I, Ljubicic V, Kirwan AF, Hood DA - PLoS ONE (2008)

Bottom Line: The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding.Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect.Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, York University, Toronto, Ontario, Canada.

ABSTRACT
The mechanisms by which PGC-1alpha gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1alpha using AICAR, an activator of AMPK, that is known to increase PGC-1alpha expression. A 2.2 kb fragment of the human PGC-1alpha promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-kappaB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1alpha promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1alpha promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1alpha promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1alpha promoter activity. The USF-1-mediated increase in PGC-1alpha promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression. This could represent a potential therapeutic target to control PGC-1alpha expression in skeletal muscle.

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Effect of GATA-4 and USF-1 overexpression on PGC-1α promoter activity and mRNA expression.A. Representative western blots of protein extracts made from C2C12 cells transfected with either 2 or 4 µg of GATA-4 or USF-1 or an empty vector (EV) control. B. USF-1 and GATA-4 were co-transfected with the pGL3 (EV; 500ng) or the p851 PGC-1α promoter reporter construct (500ng) along with the appropriate empty vector controls. Relative luciferase activities were measured 48 hours after transfection and are plotted as the fold change above empty vector. Values are means±SEM, (n = 8); * p<0.05 versus p851-EV and #, p<0.05 versus p851-USF-1 or p851-GATA-4. C. Cells were transfected with 4 µg of USF-1 or an empty vector (EV) control. EtBr-stained DNA gel of PGC-1α amplified by PCR from EV- and USF-1 transfected cells. s12rRNA was used to verify equal loading. Data are representative of one experiment with conditions repeated in duplicate (AU = arbitrary units).
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pone-0003614-g005: Effect of GATA-4 and USF-1 overexpression on PGC-1α promoter activity and mRNA expression.A. Representative western blots of protein extracts made from C2C12 cells transfected with either 2 or 4 µg of GATA-4 or USF-1 or an empty vector (EV) control. B. USF-1 and GATA-4 were co-transfected with the pGL3 (EV; 500ng) or the p851 PGC-1α promoter reporter construct (500ng) along with the appropriate empty vector controls. Relative luciferase activities were measured 48 hours after transfection and are plotted as the fold change above empty vector. Values are means±SEM, (n = 8); * p<0.05 versus p851-EV and #, p<0.05 versus p851-USF-1 or p851-GATA-4. C. Cells were transfected with 4 µg of USF-1 or an empty vector (EV) control. EtBr-stained DNA gel of PGC-1α amplified by PCR from EV- and USF-1 transfected cells. s12rRNA was used to verify equal loading. Data are representative of one experiment with conditions repeated in duplicate (AU = arbitrary units).

Mentions: We sought to identify whether GATA-4 and USF-1 could mediate PGC-1α transcription independently. Thus, we overexpressed GATA-4, USF-1 or the two in combination in muscle cells. Increases in the levels of GATA-4 or USF-1 protein were detected by Western Blotting in cells transfected with 4 µg of DNA (Fig. 5A). USF-1 overexpression alone increased promoter activity by 1.7 fold (p<0.001), in addition to producing an increase in PGC-1α mRNA (Fig. 5C). The enhanced effect of USF-1 overexpression on promoter activity was not observed when the p851mtEbox was transfected, indicating that the USF-1 effect occurs via the Ebox element (data not shown). GATA-4 overexpression led to a 2.0- fold increase in promoter activity (p<0.05; Fig. 5B), but did not lead to increases in PGC-1α mRNA. This latter result was expected, since the GATA element found within the human promoter is not conserved in the mouse. The combination of USF-1 and GATA-4 overexpression produced a further significant increase in PGC-1α promoter activity to 2.5-fold above the empty vector control. The magnitude of this increase is suggestive of an additive effect of the two proteins on PGC-1α transcription.


AMP-activated protein kinase-regulated activation of the PGC-1alpha promoter in skeletal muscle cells.

Irrcher I, Ljubicic V, Kirwan AF, Hood DA - PLoS ONE (2008)

Effect of GATA-4 and USF-1 overexpression on PGC-1α promoter activity and mRNA expression.A. Representative western blots of protein extracts made from C2C12 cells transfected with either 2 or 4 µg of GATA-4 or USF-1 or an empty vector (EV) control. B. USF-1 and GATA-4 were co-transfected with the pGL3 (EV; 500ng) or the p851 PGC-1α promoter reporter construct (500ng) along with the appropriate empty vector controls. Relative luciferase activities were measured 48 hours after transfection and are plotted as the fold change above empty vector. Values are means±SEM, (n = 8); * p<0.05 versus p851-EV and #, p<0.05 versus p851-USF-1 or p851-GATA-4. C. Cells were transfected with 4 µg of USF-1 or an empty vector (EV) control. EtBr-stained DNA gel of PGC-1α amplified by PCR from EV- and USF-1 transfected cells. s12rRNA was used to verify equal loading. Data are representative of one experiment with conditions repeated in duplicate (AU = arbitrary units).
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Related In: Results  -  Collection

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pone-0003614-g005: Effect of GATA-4 and USF-1 overexpression on PGC-1α promoter activity and mRNA expression.A. Representative western blots of protein extracts made from C2C12 cells transfected with either 2 or 4 µg of GATA-4 or USF-1 or an empty vector (EV) control. B. USF-1 and GATA-4 were co-transfected with the pGL3 (EV; 500ng) or the p851 PGC-1α promoter reporter construct (500ng) along with the appropriate empty vector controls. Relative luciferase activities were measured 48 hours after transfection and are plotted as the fold change above empty vector. Values are means±SEM, (n = 8); * p<0.05 versus p851-EV and #, p<0.05 versus p851-USF-1 or p851-GATA-4. C. Cells were transfected with 4 µg of USF-1 or an empty vector (EV) control. EtBr-stained DNA gel of PGC-1α amplified by PCR from EV- and USF-1 transfected cells. s12rRNA was used to verify equal loading. Data are representative of one experiment with conditions repeated in duplicate (AU = arbitrary units).
Mentions: We sought to identify whether GATA-4 and USF-1 could mediate PGC-1α transcription independently. Thus, we overexpressed GATA-4, USF-1 or the two in combination in muscle cells. Increases in the levels of GATA-4 or USF-1 protein were detected by Western Blotting in cells transfected with 4 µg of DNA (Fig. 5A). USF-1 overexpression alone increased promoter activity by 1.7 fold (p<0.001), in addition to producing an increase in PGC-1α mRNA (Fig. 5C). The enhanced effect of USF-1 overexpression on promoter activity was not observed when the p851mtEbox was transfected, indicating that the USF-1 effect occurs via the Ebox element (data not shown). GATA-4 overexpression led to a 2.0- fold increase in promoter activity (p<0.05; Fig. 5B), but did not lead to increases in PGC-1α mRNA. This latter result was expected, since the GATA element found within the human promoter is not conserved in the mouse. The combination of USF-1 and GATA-4 overexpression produced a further significant increase in PGC-1α promoter activity to 2.5-fold above the empty vector control. The magnitude of this increase is suggestive of an additive effect of the two proteins on PGC-1α transcription.

Bottom Line: The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding.Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect.Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, York University, Toronto, Ontario, Canada.

ABSTRACT
The mechanisms by which PGC-1alpha gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1alpha using AICAR, an activator of AMPK, that is known to increase PGC-1alpha expression. A 2.2 kb fragment of the human PGC-1alpha promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-kappaB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1alpha promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1alpha promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1alpha promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1alpha promoter activity. The USF-1-mediated increase in PGC-1alpha promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression. This could represent a potential therapeutic target to control PGC-1alpha expression in skeletal muscle.

Show MeSH
Related in: MedlinePlus