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PGC-1alpha is coupled to HIF-1alpha-dependent gene expression by increasing mitochondrial oxygen consumption in skeletal muscle cells - PubMed

  • ️Thu Jan 01 2009

PGC-1alpha is coupled to HIF-1alpha-dependent gene expression by increasing mitochondrial oxygen consumption in skeletal muscle cells

Kathleen A O'Hagan et al. Proc Natl Acad Sci U S A. 2009.

Erratum in

  • Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5449. Tambawala, Murtaza M [corrected to Tambuwala, Murtaza M]

Abstract

Mitochondrial biogenesis occurs in response to increased cellular ATP demand. The mitochondrial electron transport chain requires molecular oxygen to produce ATP. Thus, increased ATP generation after mitochondrial biogenesis results in increased oxygen demand that must be matched by a corresponding increase in oxygen supply. We found that overexpression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), which increases mitochondrial biogenesis in primary skeletal muscle cells, leads to increased expression of a cohort of genes known to be regulated by the dimeric hypoxia-inducible factor (HIF), a master regulator of the adaptive response to hypoxia. PGC-1alpha-dependent induction of HIF target genes under physiologic oxygen concentrations is not through transcriptional coactivation of HIF or up-regulation of HIF-1alpha mRNA but through HIF-1alpha protein stabilization. It occurs because of intracellular hypoxia as a result of increased oxygen consumption after mitochondrial biogenesis. Thus, we propose that at physiologic oxygen concentrations, PGC-1alpha is coupled to HIF signaling through the regulation of intracellular oxygen availability, allowing cells and tissues to match increased oxygen demand after mitochondrial biogenesis with increased oxygen supply.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Identification of HIF target gene expression in PGC-1α-overexpressing cells. (A) RT-PCR of HIF target genes in primary human skeletal muscle cells infected with either Ad-GFP or Ad-PGC-1α. (B) RT-PCR of TfR mRNA in C2C12 myotubes infected with either Ad-GFP or Ad-PGC1α. (C) TfR protein expression in L6 myotubes stably expressing PGC-1α (L6-PGC-1α) compared with control L6-laczeo (LZ) cells. β-Actin was used as a loading control. (D) RT-PCR analysis of VEGF-A and TfR in L6-PGC-1α and L6-laczeo myotubes maintained at 21% or 1% atmospheric oxygen for 24 h. *, P < 0.05; **, P < 0.01; mean ± SEM; n = 3 throughout.

Fig. 2.
Fig. 2.

Role of HIF-1α in the transactivation of the TfR promoter in cells overexpressing PGC-1α. (A) (Upper) A TfR promoter-luciferase truncation reporter series was cotransfected with PGC-1α expression vector or control vector. Luciferase activity was measured. (Lower) HRE-luciferase activity was measured in C2C12 cotransfected with PGC-1α expression vector. (B) Wild-type (pTfRB-luc; Left) or HRE-mutant TfR reporter (pTfRBmut-luc; Right) constructs were cotransfected in C2C12 myoblasts with a PGC-1α expression vector or control vector. Luciferase expression was measured after 24-h exposure to 21% or 1% O2. (C) Immunoblot analysis demonstrates siRNA silencing of HIF1-α in HeLa cells exposed to hypoxia and cotransfected with PGC-1α expression vector or a control vector. (D) HIF-1α siRNA was used to reduce HIF-1α expression in HeLa cells. Cells were cotransfected with TfR luciferase reporter and PGC-1α expression vector or control vector. Luciferase expression was measured after exposure to 21% or 1% O2 for 24 h. *, P < 0.05; **, P < 0.01; mean ± SEM; n = 3 throughout.

Fig. 3.
Fig. 3.

Effects of PGC-1α overexpression on intracellular oxygen levels. (A) (Upper) O2 concentration in L6-PGC-1α myotubes was measured by fluorescence quenching oxymetry. (Lower). Increased mitochondrial mass in L6-PGC-1α myotubes was confirmed by immunoblot analysis of cellular levels of cytochrome c. (B) C2C12 cells were transfected with either PGC-1α expression vector or control vector, and mitochondrial mass and activity was imaged with MTG (Upper) and TMRM (Lower), respectively. (Bar: 20 μm.) (C) C2C12 cells were cotransfected with near-infrared (NIR) oxygen probes, and either PGC-1α expression vector or control vector and oxygen consumption was determined. *, P < 0.05; **, P < 0.01; mean ± SEM; n = 3 throughout.

Fig. 4.
Fig. 4.

Effect of PGC-1α overexpression on HIF-1α stabilization and PHD activity. (A) Immunoblot analysis of HIF-1α in HeLa cells transfected with either PGC-1α expression vector or control vector at either 21% and 10% atmospheric O2 (Upper) or 21% and 5% atmospheric O2 (Lower). (B) HeLa cells were exposed to 21% and 5% oxygen with or without PGC-1α overepression and antimycin A (1 μg/mL), and HIF-1α levels were determined by Western blot analysis. (C) Immunoblot analysis of ODD-GFP-V5 expression in HeLa cells transfected with either PGC-1α expression vector or control vector at either 21% or 10% atmospheric O2. (D) Immunoblot analysis of ODD-GFP-V5 expression in C2C12 cells transfected with either PGC-1α expression vector or control vector in 21% or 10% O2. n = 3 throughout.

Fig. 5.
Fig. 5.

Schematic representation of HIF target gene expression by PGC-1α. PGC-1α expression results in mitochondrial biogenesis and increased oxygen consumption. The resultant drop in intracellular pO2 levels leads to decreased HIF hydroxylase activity and HIF stabilization with resultant gene expression that facilitates an increase in oxygen supply.

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