The estrogen-related receptor alpha (ERRalpha) functions in PPARgamma coactivator 1alpha (PGC-1alpha)-induced mitochondrial biogenesis

Abstract

Estrogen-related receptor alpha (ERRalpha) is one of the first orphan nuclear receptors to be identified, yet its physiological functions are still unclear. We show here that ERRalpha is an effector of the transcriptional coactivator PGC-1alpha [peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha], and that it regulates the expression of genes involved in oxidative phosphorylation and mitochondrial biogenesis. Inhibition of ERRalpha compromises the ability of PGC-1alpha to induce the expression of genes encoding mitochondrial proteins and to increase mitochondrial DNA content. A constitutively active form of ERRalpha is sufficient to elicit both responses. ERRalpha binding sites are present in the transcriptional control regions of ERRalpha/PGC-1alpha-induced genes and contribute to the transcriptional response to PGC-1alpha. The ERRalpha-regulated genes described here have been reported to be expressed at reduced levels in humans that are insulin-resistant. Thus, changes in ERRalpha activity could be linked to pathological changes in metabolic disease, such as diabetes.

Fig. 1.

PGC-1α induces mitochondrial biogenesis in SAOS2 cells, dependent on interaction with nuclear receptors. Cells were infected with GFP- or (WT or mutant L2/3A) PGC-1α-expressing adenoviruses at an moi of 40. (A) Mitochondria in cells labeled with CM-H₂Xros were imaged 48 h after infection. (B) Accumulation of MitoFluor Red 594 in cells was measured by flow cytometry 48 h after infection. Gm represents the geometric mean fluorescence intensity of 20,000 cells. (C) Mitochondrial (COX2) DNA levels normalized to nuclear (β actin) DNA levels are expressed relative to levels in control cells expressing GFP, which were set to 1, at 48 and 60 h after infection. Data are the mean ± SEM of three experiments performed in duplicates. *, P < 0.0001 versus WT PGC-1α at 48 h; **, P < 0.001 versus WT PGC-1α at 60 h, as determined by the Student's t test. (D) mRNA levels of ATPsynβ, Cyt c, and COX4 at 48 h after infection were determined by quantitative RT-PCR, normalized to the mRNA levels of 36B4, and expressed relative to levels in GFP-infected cells. (E) Protein levels of PGC-1α and ERRα were determined by Western analysis at 48 h after infection.

Fig. 2.

Inhibition of ERRα expression impairs the induction of mitochondrial biogenesis by PGC-1α. SAOS2 cells were infected with control-(AdSUPER) or an adenovirus-expressing siRNA for ERRα (siERRα) at a moi of 100. Two days later, cells were infected with GFP- or PGC-1α-expressing adenoviruses at a moi of 20 (A and B) or 40 (C). Cells were harvested 24 h (A and B) or 48 h (C) later. (A) ERRα mRNA levels were determined by quantitative RT-PCR and normalized to 36B4 levels. Data shown are the mean ± SEM of three experiments performed in duplicates. (B) mRNA levels for mtTFA, Tim22, IDH3A, ATPsynβ, carnitine/acylcarnitine translocase (CACT), Cyt c, NRF-1, and NRF-2, were determined by quantitative RT-PCR, normalized to the mRNA levels of 36B4, and expressed relative to levels in AdSUPER/GFP infected cells. Data are the mean ± SEM of three experiments performed in duplicates. *, P < 0.02; **, P < 0.003; ***, P < 0.0005 versus PGC-1α-expressing cells in the absence of siERRα. (C) Mitochondrial (COX2) DNA levels were normalized to nuclear (β actin) DNA levels and expressed relative to levels in control-infected (AdSU-PER and GFP) cells, which were set to 1. Data are the mean ± SEM of two experiments performed in duplicates. *, P < 0.008 versus PGC-1α-expressing cells in the absence of siERRα.

Fig. 3.

A constitutively active ERRα induces mitochondrial biogenesis. SAOS2 cells were infected with adenoviruses expressing GFP, PGC-1α, ERRα, VP16, or VP16-ERRα (moi 40) and analyzed 24 h (A) or 60 h (B) later. (A) mRNA levels for the indicated genes were determined by quantitative RT-PCR, as in Fig. 1D. Data are the mean ± SEM of three experiments performed in duplicates. *, P < 0.002; **, P ≤ 0.0001; ***, P < 0.0004 versus GFP-infected cells. (B) Mitochondrial DNA content was determined as in Fig. 1C. Data are the mean ± SEM of two experiments performed in triplicates. *, P < 0.0001 versus GFP-infected cells.

Fig. 4.

ERRα recognizes sites in ATPsynβ and Cyt c regulatory sequences. (A) Sequences of candidate ERREs identified by in silico analysis. The TRα/-443 ERRE has been described (25). (B) Electrophoretic mobility shift assay. ERRα was incubated with a ³²P-labeled oligonucleotide containing the ERRE of Cyt c/-596 in the presence of unlabeled oligonucleotides as indicated. M4 oligonucleotide has the TRα/-443 sequence with a 2-bp substitution in the core ERRE (25).

Fig. 5.

The ERREs of ATPsynβ and Cyt c contribute to the transcriptional response to PGC-1α. COS7 cells were transfected with reporters pATPsynβ/-385Luc or pCytc/-686Luc, [WT and bearing mutations (mt) or deletions (Δ) at the ERREs and NRF-1/NRF-2 binding sites] and control vector (-), PGC-1α-, and/or ERRα-expressing plasmids as indicated. Data are expressed as fold activation by PGC-1α or PGC-1α/ERRα, with the basal activity of each construct (white bars) set to 1, and are the mean ± SEM of at least three experiments performed in duplicates.