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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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AMPK activation induces PGC-1α mRNA expression and transcriptionally activates the PGC-1α promoter.C2C12 cells were treated with either AICAR (1 mM) or Vehicle for 24 hrs. A. Representative Western Blot probed with a Phospho-AMPKα (Thr172), stripped and then re-probed with total AMPKα for loading control (upper panel). Summary of repeated experiments of the effect of AICAR on AMPK activation is shown (lower panel; n = 4). B. upper panel, EtBr-stained DNA gel of PGC-1α amplified by PCR from vehicle- and AICAR-treated cells. GAPDH was also amplified by PCR and used to verify equal loading. Lower panel: A summary of repeated experiments of the effects of AICAR on PGC-1α mRNA expression (n = 3). C. AICAR-induced transcriptional regulation of the PGC-1α promoter. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown (n = 4–6). D. The AICAR-responsive region (ARR) from −473 to −821 was cloned into the pGL4.23 minimal promoter vector and AICAR-induced transcriptional regulation of this region was assessed (n = 3). For all data, values are means±S.E.M, *, p<0.05 versus Vehicle-treated control; §, p<005 versus pGL4.23.
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pone-0003614-g002: AMPK activation induces PGC-1α mRNA expression and transcriptionally activates the PGC-1α promoter.C2C12 cells were treated with either AICAR (1 mM) or Vehicle for 24 hrs. A. Representative Western Blot probed with a Phospho-AMPKα (Thr172), stripped and then re-probed with total AMPKα for loading control (upper panel). Summary of repeated experiments of the effect of AICAR on AMPK activation is shown (lower panel; n = 4). B. upper panel, EtBr-stained DNA gel of PGC-1α amplified by PCR from vehicle- and AICAR-treated cells. GAPDH was also amplified by PCR and used to verify equal loading. Lower panel: A summary of repeated experiments of the effects of AICAR on PGC-1α mRNA expression (n = 3). C. AICAR-induced transcriptional regulation of the PGC-1α promoter. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown (n = 4–6). D. The AICAR-responsive region (ARR) from −473 to −821 was cloned into the pGL4.23 minimal promoter vector and AICAR-induced transcriptional regulation of this region was assessed (n = 3). For all data, values are means±S.E.M, *, p<0.05 versus Vehicle-treated control; §, p<005 versus pGL4.23.

Mentions: The nucleotide sequence +28 to −2190 corresponding to the proximal 2-kb hPGC-1α promoter is shown. The arrows indicate the transcription start sites, which have been previously described [14]. Putative binding sites for transcription factors are either underlined or overlined. Also included are binding sites for transcription factors that have previously been characterized [12;13;19]. Numbers enclosed in circles represent the 5′- deletions of the PGC-1α promoter reporter constructs p 2215, p 1164, p 851, p501, p 191 as shown in Fig. 2.


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)

AMPK activation induces PGC-1α mRNA expression and transcriptionally activates the PGC-1α promoter.C2C12 cells were treated with either AICAR (1 mM) or Vehicle for 24 hrs. A. Representative Western Blot probed with a Phospho-AMPKα (Thr172), stripped and then re-probed with total AMPKα for loading control (upper panel). Summary of repeated experiments of the effect of AICAR on AMPK activation is shown (lower panel; n = 4). B. upper panel, EtBr-stained DNA gel of PGC-1α amplified by PCR from vehicle- and AICAR-treated cells. GAPDH was also amplified by PCR and used to verify equal loading. Lower panel: A summary of repeated experiments of the effects of AICAR on PGC-1α mRNA expression (n = 3). C. AICAR-induced transcriptional regulation of the PGC-1α promoter. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown (n = 4–6). D. The AICAR-responsive region (ARR) from −473 to −821 was cloned into the pGL4.23 minimal promoter vector and AICAR-induced transcriptional regulation of this region was assessed (n = 3). For all data, values are means±S.E.M, *, p<0.05 versus Vehicle-treated control; §, p<005 versus pGL4.23.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2570798&req=5

pone-0003614-g002: AMPK activation induces PGC-1α mRNA expression and transcriptionally activates the PGC-1α promoter.C2C12 cells were treated with either AICAR (1 mM) or Vehicle for 24 hrs. A. Representative Western Blot probed with a Phospho-AMPKα (Thr172), stripped and then re-probed with total AMPKα for loading control (upper panel). Summary of repeated experiments of the effect of AICAR on AMPK activation is shown (lower panel; n = 4). B. upper panel, EtBr-stained DNA gel of PGC-1α amplified by PCR from vehicle- and AICAR-treated cells. GAPDH was also amplified by PCR and used to verify equal loading. Lower panel: A summary of repeated experiments of the effects of AICAR on PGC-1α mRNA expression (n = 3). C. AICAR-induced transcriptional regulation of the PGC-1α promoter. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown (n = 4–6). D. The AICAR-responsive region (ARR) from −473 to −821 was cloned into the pGL4.23 minimal promoter vector and AICAR-induced transcriptional regulation of this region was assessed (n = 3). For all data, values are means±S.E.M, *, p<0.05 versus Vehicle-treated control; §, p<005 versus pGL4.23.
Mentions: The nucleotide sequence +28 to −2190 corresponding to the proximal 2-kb hPGC-1α promoter is shown. The arrows indicate the transcription start sites, which have been previously described [14]. Putative binding sites for transcription factors are either underlined or overlined. Also included are binding sites for transcription factors that have previously been characterized [12;13;19]. Numbers enclosed in circles represent the 5′- deletions of the PGC-1α promoter reporter constructs p 2215, p 1164, p 851, p501, p 191 as shown in Fig. 2.

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