the co-activator pgc-1: from flies to mammals pgc-1 co...
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THE CO-ACTIVATOR PGC-1: from flies to mammals
PGC-1 co-activator family regulates muscle formation and mitochondrial
biogenesis
Mª Cruz Marín, Juan J. Arredondo & Rafael Garesse and Margarita Cervera*
Instituto Investigaciones Biomédicas Alberto Sols. Facultad de Medicina, UAM-CSIC
Running title: Drosophila co-activator PGC-1
*Address correspondence to: Margarita Cervera, Instituto Investigaciones Biomédicas
Alberto Sols. Facultad de Medicina, UAM-CSIC. Arzobispo Morcillo 4, 28029 Madrid,
SPAIN. Fax: +34 91 585 4401 Phone: +34 91 497 5402; email:
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ABSTRACT
Transcriptional co-activators and co-repressors are emerging as physiological
integrators of energy metabolism and other biological processes. Tissues and organs
need to adapt to different critical physiological process as energetic metabolism,
apoptosis or ROS signaling. The correct number of structural and functionally
differentiated mitochondria mediates all these processes. In consequence, mitochondrial
biogenesis must be strictly regulated, a process that implies the coordinated regulation
of nuclear and mitochondrial genome gene expression. In mammals, PGC-1 co-
activators are essential to integrate the response of a network of specific transcription
factors involved in mitochondria biogenesis. Here, we focus on the role of the PGC-1
co-activator family. These proteins are key players in the control of organ-specific
biologic responses to the physiologic environment. Emphasis will be given to tissue-
specific regulatory features relevant to muscle. Our knowledge of the PGC-1 family in
vertebrates has augmented, but even now the molecular networks on which these co-
regulators are implicated, the PGC-1 response to the external signal and how molecular
pathways are functionally integrated to activate gene expression programs are mostly
unknown. In this sense, the studies of the function of the unique member of Drosophila
identified and the comparative analysis between flies and mammals will help in our
understanding of the integrative signals mediated by PGC-1 co-activator protein family.
Here we show that Drosophila PGC-1, Spargel/dPGC-1 may play also a crucial role in
mitochondrial biogenesis. Homozygous dPGC-1 null mutants are not fertile and present
strong muscle phenotype. These mutants are unable to fly and electron microscopy
studies of flight muscles from these animals show clear fiber degeneration. Very
interestingly, we have also found a strong reduction of both mitochondrial electron
density and size. Fly and mammals comparative analysis will be presented.
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INTRODUCTION
During development as animal grow, cells need to adapt to different physiological
process as energetic metabolism, apoptosis or ROS signaling. In these processes,
mitochondria have critical anabolic and catabolic functions in metabolizing nutrients
and in adapting their cellular physiology. The correct number of structural and
functionally differentiated mitochondria mediates all these actions. Therefore,
mitochondrial biogenesis must be strictly regulated and nuclear and mitochondrial
genome’s gene expression must be coordinated.
The last two decades have witnessed a tremendous expansion in our knowledge of the
mechanisms employed by eukaryotic cells to control gene activity. Transcriptional co-
activators and co-repressors are emerging as physiological integrators of energy
metabolism and other biological events [1, 2]. A common theme of these co-activator
regulatory networks it that, by co-activating multiple transcription factor targets they are
able to coordinate diverse biological processes that constitute biological responses [3,
4].
To learn about mitochondrial biogenesis, most studies focused on individual tissues that
show an enormous increase in mitochondrial activity and mass in response to external
stimuli, for example, during brown adipose tissue, muscle or perinatal heart maturation.
These studies led to identification and characterization of the PGC-1 family of
transcriptional co-activators that are potent inducers of mitochondrial biogenesis [5, 6].
A critical aspect of the PGC-1 co-activators is that they are highly versatile and have the
ability to interact with many different transcription factors. In fact, in vertebrates, the
PGC-1 proteins are inducible transcriptional co-activators orchestrating control of
cellular energy metabolism [4, 7]. Less is known about the role of the unique member of
the PGC-1 family present in the Drosophila model system, recently identify by our
group and others [8].
Although our knowledge of the PGC-1α in vertebrates has increased substantially, not
all the molecular pathways in the biological processes on which these co-regulators are
involved are known. Moreover, it is unclear how PGC-1α responds to the different
external stimuli and how molecular pathways are functionally integrated in different
gene expression programs.
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In flies, the homologue of PGC-1a, Spargel/dPGC-1, seems to play a critical role in
mitochondrial biogenesis in fat body and muscle, coordinating mitochondrial activity
and insulin signaling [8]. Drosophila is an excellent system to profound the knowledge
of mitochondria and muscle for three reasons: 1) Its powerful genetics combined with a
wealth of available cell and molecular biology techniques; 2) Genetic pathways that
guide basic biological processes, in Drosophila and in vertebrates, have remained
largely intact throughout evolution. This conservation enables rapid analysis of these
processes in vertebrates and 3) Habitually in flies, one single member represents a
functional gene family in vertebrates facilitating functional studies.
PGC-1 family members in vertebrates drive mitochondrial biogenesis through co-
activation of nuclear transcription factors. The majority of these factors are conserved in
flies. Thus, the transcription factors, Erect wing, homolog of NRF1, plays a critical role
in neural and muscle development; Delg, homolog of NRF2, is required during
oogenesis; NRGF, binds NRG sequences (nuclear regulatory gene elements) localized
in mitochondrial gene promoters and DREF is involved in DNA replication and cell
cycle control are conserved [8, 9]. At the moment, no Drosophila transcriptional co-
regulators have been characterized as physiological integrators of energy metabolism
and other biological processes. We think flies will help, in the near future, to unravel
some of the open and common questions about the exact role of PGC-1 in the
integration and coordination of the different regulatory networks in which this co-
activator is implicated.
This review will focus on the role of the PGC-1 co-activator family. These proteins are
key players in the control of organ-specific biologic responses to the physiologic milieu.
Emphasis will be given to tissue-specific regulatory features relevant to fat body and
muscle. Our knowledge of the PGC-1family in vertebrates has augmented, but even
now the molecular networks on which these co-regulators are implicated are largely
known. At the moment, the complete mechanisms of how PGC-1 responds to the
external signals and how molecular pathways are functionally integrated to activate
gene expression programs are unknown. In this sense, the studies of the function of the
unique member of Drosophila identified [8], and the comparative analysis between flies
and mammals will help in our understanding of the integrative signals mediated by
PGC-1 co-activator protein family. Fly and mammals comparative analysis will be
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presented.
RESULTS AND DISCUSSION
Molecular Conservation of PGC-1 co-activator family genes
In silico analysis has shown that PGC-1 family of co-activators is conserved in many
chordate species, including primates, rodents, ruminants, birds, amphibians and fishes
[5]. In vertebrates, three members compose this family; the first identified was PGC-
1α , characterized as a PPARγ -interacting protein and isolated from brown fat [10].
PGC-1α has two structural homologues, PGC-1β which shares an extensive sequence
homology with PGC-1α [11, 12], and PRC-related co-activator (PRC) with more
limited overall sequence similarity with PGC1α and PGC1β but sharing the key
structural features that define the family [13]. Detailed protein analysis reveals different
conserved functional domains (Figure 1). In the N-terminal acidic region, there is a
transcription activation domain that contains one nuclear hormone co-activator
signature motif (LXXLL) in PGC-1α and two of these motifs in PGC-1β that are crucial
for co-activation through certain nuclear receptors. In the central region of the PGC-1
proteins, a regulatory domain, mainly involved in co-activation through binding to
different mitochondrial transcriptional factors, exists. In the C-terminal region a
Ser/Arg-rich motif is situated close to a RNA binding domain [5].
In Drosophila melanogaster, a single homologue, annotated CG9809 and located in
chromosome 3R, has been identified [14]. This gene encodes a predicted protein of
1.088 amino acids and contains a RNA binding motif in the C-terminal region that
exhibits approximately 60% of homology with the same region in mammalian PGC-1 α
and β genes. Moreover, the Drosophila protein includes an acidic NH2-terminal
domain and an arginine-serine-rich domain common with its mammalian homologue
genes [14, 15] (Figure 1). Although the canonical LXXLL nuclear receptor binding
motif is not present, dPGC-1 contains a conserved variant of this motif FXXLL in the
C-terminal region which has been demonstrated to be involved in nuclear receptor
binding [16]. Thus, Drosophila and vertebrate PGC-1α proteins contain the functional
domain involved in nuclear receptor binding.
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FIGURE 1: Molecular conservation of mammalian and Drosophila PGC-1 co-activator family. In
the upper part the sequence homology of PRC, PGC-1β and PGC-1α proteins is shown. Color boxes
represent protein domains: activation domain (red), Arg/Ser rich domain (orange) and RNA binding
domain (blue). Black lines denote LXXL motifs (nuclear receptor binding sites). In the lower part, PGC-
1α and PGC-1 of Drosophila (dPGC-1) sequence homology is presented. Note the high percentage of
homology in RNA binding domain, RRM domain. No LXXL-like motifs have been identified in
Drosophila. Also note that sequence F(D/E)XLL is common to the carboxy-terminal domains of all PGC-
1 family members.
PGC1 proteins function as inducible transcriptional co-activators: molecular mechanism
The discovery of the first transcriptional co-activators and co-repressors for nuclear
hormone receptors and other transcription factors initiated a novel concept centered
around the control of specific genetic programs coordinated at the level of these proteins
[10]. Co-activators act interacting with various transcription factors, allowing for the
coordinated expression of gene sets in response to specific signals. Another important
and interesting implication of co-activators is the possibility to activate gene expression
in very specific contexts. The members of the inducible PGC-1 transcriptional co-
activator family posses a common function in mitochondrial physiology in addition to
control other specific programs. In the cells, PGC-1 gene expression is modified in
response to a variety of extracellular stimuli including temperature, energy deprivation
and availability of nutrients and grows factors [6, 17, 18]. However, these co-activators
exhibit differential specificities for transcription factors utilization, which in turn
modulates the physiological response orchestrated by each co-activator [7].
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The molecular mechanisms through which PGC-1α activates gene expression are
poorly understood. These co-activators form part of protein complexes whose concrete
protein identity and enzymatic activities are not completely known. Molecular studies,
mostly performed in cell cultures, indicate that PGC-1α and PGC-1β function through
recruiting chromatin-remodeling complexes that include those involved in histone
modification and nucleosome remodeling (reviewed in [18]). PGC-1α activates
transcription by physical interaction with p300 histone acetyltransferase domain
containing proteins as P300, CBP, SRC-1 and GCN5 as well as the mediator protein
complex, which is thought to recruit RNA polymerase II [19-22]. In the liver, where it
participates in the regulation of lipid metabolism, PGC-1α interacts with BRG1/BRM-
associated factor (BAF60a), a subunit of SW1/SNF nucleosome remodeling complex.
This interaction is required for the transcriptional function of PPARα (peroxisome
proliferator-activated receptor) and may be implicated in transcriptional function of
other nuclear hormone receptors [23, 24]. The recruitment of these histone acetyl-
transferase complexes containing PGC-1α to the proximity of the target-genes increases
histone acetylation, one of the first steps in transcriptional activation.
Another important aspect to understand is how these complexes may be spatially and
temporally assembled with PGC-1α to modulate and activate gene expression. Not
much is known about how these events occur, but modulation of transcription is
achieved through acetylation /deacetylation of PGC-1α itself. PGC-1 α has been found
in association with two additional different transcriptional complexes, the GCN5 and
TIP60 acetyl transferase complexes. The acetyl transferase GCN5 directly acetylates
PGC-1α at multiple lysine residues and the acetylated protein negatively regulates its
transcriptional activation function, at least in part through nuclear sub-localization [21].
The opposite role is played by lysine de-acetylation of PGC-1 by SIRT1 which
increases its function in response to cellular nutrients [25-28].
The model described above is perhaps simplistic and does not take into account other
additional PGC-1 post-translational modifications such as phosphorylation and
methylation whose precise functions are still unknown. Thus, PGC-1α phosphorylation
by the p38 MAPK and AMP kinase (AMPK) has been described [29-31]. Also,
methylation [32] and the novel O-linked β-N-acetylglucosamine (O-GlcNAc)
modifications on PGC-1α have been shown. The last, modulating the activity of FoxO
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transcription factors in liver culture cells [33]. On the other hand, the RNA-binding
domain of PGC-1α has been implicated in processing many mRNAs whose
transcription it initiates [34].
PCG-1α, PGC-1β and PRC act as transcriptional co-activators and function through
direct physical interaction with transcription factors directly bound to DNA promoter
regions. Thus, through their N-terminal region they interact with different hormone
nuclear receptors including PPARs, HNF4α, GR and ERRα while through their central
region bind transcription factors implicated in the activation of mitochondrial
biogenesis and respiratory function, such as NRF1, NRF2, TFAM, TBPs and MEF2C
[5, 7]. In addition, they interact with the RNA-processing C-terminal region with other
transcriptional factor group such as FoxO and YY1 involved in other pathways [35]. As
mentioned in the Introduction, the ability of PGC-1α to interact with different
transcription factors allows for the coordinated expression of gene sets in response to
specific signals (Figure 2).
In this context, in vertebrates, it has been described that NRF-1 transcription factor
regulates the expression of many genes involved in mitochondrial regulatory including
the majority of nuclear genes that encode subunits of the five respiratory complexes as
well as the expression of Tfam [36], and the two TFB genes [37], the main regulators of
mitochondrial transcription. Both NRF-1 and PGC-1α are up-regulated during adaptive
mitochondrial response of skeletal muscle to exercise training [38, 39] and in cultured
myotubes in response to increased calcium [40]. PGC-1β also is a potent co-activator of
NRF-1. An increase in PGC-1β expression in transgenic mice promotes, associated
with an increase of oxidative muscle fibers, a massive increase of mitochondrial
contend and high levels of mitochondrial respiratory genes transcription [41]. Thus,
PGC-1α , PGC-1β and PRC bind NRF-1 in order to trans-activate NRF-1 target genes.
Serum treatment of quiescent fibroblast induces PRC expression, followed by an
increase of TFAM, TFB1M and B2 genes, as well as nuclear and mitochondrial
encoded subunits of respiratory complexes. Functional binding sites for NRF-2 (the
human homolog of mouse GA- binding protein) have been localized to the promoters of
numerous genes related to respiratory chain, mainly COX subunits [42, 43], as well as
to TFAM [44],TFB1 and B2 promoters [37]. It has been speculated that coordinated
regulation of both NRF-1 and NRF-2 proteins mediated by PGC-1 family members
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could be a mechanism to ensure the correct expression of essential mitochondrial
proteins.
In the same context, the hormone nuclear receptor ERRα levels are high in oxidative
tissues and it modulates the activation of MCAD (medium-chain acyl-coenzyme A
deshydrogenase) promoter in the β -oxidation pathway [45]. ERRα has also been
implicated in PGC-1 induced mitochondrial biogenesis. Mootha and col. (2004),
through a computational approach to detect cis-regulatory motifs coupled with PGC-
1α -induced genome-wide transcriptional profiles, identified ERRα and GA-binding
protein α (NRF-2 α ) as key transcription factors regulating the OXPHOS pathway.
Cellular assays confirmed that both proteins were PGC-1α partners in muscle,
constituting a double-positive-feedback loop that drives the expression of many
OXPHOS genes [46].
FIGURE 2: Illustration summarizing PGC-1 functions in mammalian muscle cells. PGC-1 co-
activator integrates various physiological cues (red arrow) that are transduced into different signaling
cascades (yellow arrow) modulating the levels of PGC-1 mRNA and/or protein. In turn, PGC-1 mediated
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co-activation of several nuclear transcription factors orchestrates specific responses into the muscle cell
(green arrow).
In addition to its role in respiratory chain genes and mitochondrial transcription, PGC-
1α promotes mitochondrial oxidative functions by inducing the expression of genes of
the mitochondrial fatty acid oxidation and heme biosynthetic pathways. Ligand-
dependent binding PPARα to PGC-1α induce the trans-activation of PPARα target
genes [47]. Over-expression of PGC-1α induces MCAD gene expression through its
interaction with ERRα [45] and together with NRF-1 and FOXO1 (forkhead, box
family member) induces δ-aminolevulinate synthase, a critical enzyme in the heme
biosynthetic pathway [48]. Previously, it has been showed that control of glucose
oxidation through pyruvate deshydrogenase complex (PDC) could be regulated by
PGC-1α . PGC-1α over-expression induces, via ERRα , the transcriptional up-
regulation of PDK-4 (pyruvate deshydrogenase kinase), which inactivates PDC by
phosphorylation. In addition, PGC-1α over-expression effects were lost in MEFs
derived from a ERRα null mice [49]. Recent studies showed that PDK4 gene
expression is stimulated by thyroid hormone T(3) demonstrating that following T(3)
administration there is an increase in the association of PGC-1 α with the PDK4
promoter [50].
In flies, the majority of the transcription factors that interact with mammalian PCG-1α,
PGC1β and PRC have been identified. Thus, Drosophila transcription factor erect wing
(ewg) is the molecular homologue of the NRF-1 gene of vertebrates. ewg has been
reported as a critical transcription factor during muscle and nervous system
development. Surprisingly, to date, no ewg function related to mitochondrial biogenesis
or OXPHOS respiratory chain has been described [51]. Recently, Haussmann and col.
(2008) have showed that EWG neuronal expression restricts synaptic growth at
neuromuscular junctions. Using a functional genomics approach in late embryos they
demonstrated that EWG acts increasing the mRNA levels of genes involved in
transcriptional and post-transcriptional regulation of gene expression indicating the
existence of an extensive regulatory network. Then, EWG restricts synaptic growth by
integrating multiple cellular signaling pathways into an regulatory gene expression
network [52]. This result may be accounted with the role of NRF-1 as a critical
regulator in vertebrates. However, genes differentially regulated in ewg mutants were
not enriched in mitochondrial genes or genes involved in mitochondrial biogenesis.
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Also, mitochondrial content in synapses was not affected in these mutants. But
otherwise, in ewg mutants several genes that are differentially regulated are involved in
energy metabolism through the generation of ATP by inhibiting gluconeogenesis and
mobilizing lipids stores [52]. Preliminary studies carried out in our lab show that the
transcription of ewg gene is reduced in null flies. These results are in accordance to the
muscle phenotype observed in this null mutant (unpublished results). Since, PGC-1α
null mice have low levels of transcription of NMJ genes in nervous system [53], it is
worth to speculate that ewg role in NMJ transcriptional regulation in Drosophila could
be could be mediated by dPGC-1.
NRF2/GABPα in Drosophila, named Delg, is required in oogenesis and seems to have
an equivalent function to its mammalian homologue [54]. This factor is involved in the
transcriptional regulation of several mitochondrial genes. In addition, is required to
adjust mitochondrial mass depending on nutrients availability [9]. Previous studies
showed that dPGC-1 and Delg have a common role in regulating the expression of
many genes encoding mitochondrial proteins in mid-third instar larvae fat body.
Moreover dPGC-1 and Delg show synergistic effects over mitochondrial content as
demonstrated dPGC/Delg double mutant [8].
Additional Drosophila transcription factors as DREF [DRE (DNA replication-related
element)-binding factor] regulate the transcription a group of genes involved in
mitochondrial DNA replication and maintenance as well as several cell proliferation-
related genes [55]. NRGF (nuclear respiratory gene) is a transcription factor that
regulates the expression of genes with mitochondrial function. Among them, are found
subunits of the electron transport chain, proteins involved in the oxidative metabolism
and mitochondrial biogenesis. NRGF binds the NRG response elements which are
found in more than 50% of the promoters of these genes [56]. Currently, it has been
speculated that this factor could be putative co-regulators of dPGC-1 mediated
response. Future approaches will allow the identification of novel co-regulators
indispensable to determine molecular mechanism of dPGC-1 in Drosophila.
Physiological pathways implicated in external response to modulate PGC-1 expression
In response to external stimuli, physiological effectors pathways mediate changes in the
transcriptional expression of PGC-1 co-activators. The c-AMP-dependent pathway is
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one of several that direct the induction of PGC-1α in a number of tissues. c-AMP is
induced in response to thermal or fasting signals so that, c-AMP high levels lead to
activation of CREB family members, through its phosphorylation by protein kinase A
(PKA). The PGC-1α promoter has a potent c-AMP response element (CRE) that serves
as a target for CREB- mediated transcription activation. During energy deprivation
conditions (reduced ATP) such as starvation, ischemia and chronic metabolic stress,
AMP-activated protein kinase (AMPK) serves as an energy sensor for whole body
energy regulation [57]. AMPK is activated by decreases in both the ATP/AMP ratio and
phosphocreatine content. After activation it increases glucose transport, fatty acid
oxidation, and mitochondrial biogenesis. In skeletal muscle AMPK directly
phosphorylates PGC-1α and increases its expression through a feedback loop [31, 58].
Also, skeletal muscle adapts to chronic physical activity through the
calcium/calmodulin-dependent protein kinase IV (CaMKIV) and calcineurin A (CnA).
PGC-1α promoter is regulated by both CaMKIV and CnA activity, and activates PGC-
1α through the binding of c-AMP response element-binding. Furthermore, the calcium-
signaling pathway may result in a stable induction of PGC-1α , contributing to the
relatively stable nature of muscle fiber-type determination [59].
PGC-1α also is induced via cGMP-dependent signaling resulted from elevates levels of
nitric oxide (NO). Long-term exposure of cells in culture to low concentrations of NO
induces mitochondrial biogenesis. This process is mediated by cGMP, resulting from
NO-dependent activation of soluble guanylatecyclase (sGC), and involves increased
expression of PGC-1α , NRF-1 and Tfam [60]. In addition, studies in mice deficient in
endothelial NO synthase (eNOS) present reduced mitochondrial biogenesis in tissues
associated with reduced energy expenditure. NO/cGMP-dependent mitochondrial
biogenesis yields functionally active mitochondria, in terms of respiratory function and
metabolic activity [61].
Almost nothing is known in Drosophila about how dPGC-1 responds to the different
external stimuli and how molecular pathways are functionally integrated. Recently
microarray studies have shown that starvation, produces a subsequent reduction of
insulin pathway and a decrease of expression of genes encoding mitochondrial proteins
[14, 62]. The over-expression of insulin receptor (ISR-1) induces an increase of dPGC-1
transcription; additionally, transcriptional changes, produced in response to IRS-1
signaling, is attenuated in hypomorphicdPGC-1 mutant suggesting that dPGC-1 is a
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mediator of insulin signaling [8]. In other respect, which transcription factors are co-
activated by dPGC-1 to integrate different signal also remain unidentified.
Tissue protein levels and functions of PGC-1 co-activators
High levels of PGC-1 co-activators are detected in those tissues with high metabolic
activity including brown adipose tissue, liver, brain, heart and skeletal muscle. In
contrast with other transcriptional co-activators, PGC-1 proteins are highly induced in
response to developmental stage-specific and physiological cues [6, 17, 18]. PGC-1α
and PGC-1β are induced by cold exposure, exercise and fasting in tissue-specific
patterns [10, 39, 63, 64]. In addition, PGC-1α is induced in heart at birth and during
postnatal stages. These stages involve a great mitochondrial biogenesis response as well
as, in parallel, high energetic requirement [65-67]. In the case of Drosophila dPGC-1
the situation is comparable. The protein is expressed ubiquitously in flies but high levels
are detected in the same tissues than in vertebrates, such as skeletal and somatic
muscles, fat body, lymph gland and central nervous system
(http://insitu.fruitfly.org/cgi-bin/ex/insitu.pl). Curiously, high levels of mRNA are
detected also in oocytes, showing that dPGC-1 is supplied like maternal deposit [68].
Cells with different functions and energetic requirements have different mitochondrial
content, i.e. number of mitochondria, mtDNA content and/or structures. In addition,
cells may adapt mitochondrial function in response to cellular or environmental signals
such as hormones, grow factors, changes in temperature, physiological activity or
developmental cues. So, the control of mitochondrial biogenesis and function implies
complex and integrated regulatory networks that depend on the coordinated expression
of two genomes located in different sub-cellular compartments, the nucleus and the
mitochondria [69-71]. PGC-1 family members play a critical role in this inter-genomic
communication reviewed in [7].
First of all, in this situation, an important question to answer is if PGC1α co-activator is
indispensable for basal mitochondrial physiology. Studies of PGC-1α loss-of-function
in mice indicate that this co-activator is dispensable for the basal physiologic processes
of mitochondria metabolism or fetal development. However, these experiments also
demonstrated that mice have severely prejudiced their ability to respond to external
stimuli such as stress, cold exposure or starvation [72, 73]. In this context, therefore,
PGC-1a seems to be a molecular switch of the metabolic variation to external stimuli.
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PGC-1a not only plays an essential role in regulating energetic metabolism but also in
many other processes and events in which mitochondria plays a fundamental role.
The physiological significance of PGC-1α and PGC-1β in mitochondrial energy
metabolism has been studied by several approaches. Studies of over-expression in
mammalian culture cell or transgenic mouse, have demonstrated that both PGC-1α or
PGC-1β are able to activate, both, the expression of a number of genes important for
mitochondrial biogenesis, and respiratory function in adipocytes, cardiac myocytes and
myogenic cells [11, 30, 65, 74, 75]. These properties are shared by PGC-1β. PGC-1α
deficiency profoundly perturbs contractile function and leads heart failure [66]. In
human, the expression of PGC-1 and mitochondrial OXPHOS genes is significantly
reduced in skeletal muscle of diabetic patients, implicating a potential role for this
factor in the pathogenesis of insulin resistance [46]. Recently, studies in vivo and in
vitro demonstrated that PGC-1α stimulates a broad and powerful program of NMJ
(neuromuscular junctions)-linked gene expression in skeletal muscle [53]. They
concluded that PGC-1α to be a key integrator of neuromuscular activity in skeletal
muscle. Ectopic expression of PGC-1α in muscle results in increased mitochondrial
number and function as well as an increase in oxidative, fatigue-resistant muscle fibers.
PGC-1 co-activators mediate muscle external adaptive stimuli (Figure 2). Whole body
PGC-1α knock-out mice have a very complex phenotype but do not have a marked
skeletal muscle phenotype. They thus analyzed skeletal muscle-specific PGC-1αknock-
out mice to identify a specific role for PGC-1α in skeletal muscle function. These mice
exhibit a shift from oxidative type I and IIa toward type IIx and IIb muscle fibers.
Moreover, skeletal muscle-specific PGC-1αknock-out animals have reduced endurance
capacity and exhibit fiber damage and elevated markers of inflammation following
treadmill running. Their data demonstrate a critical role for PGC-1α in maintenance of
normal fiber type composition and of muscle fiber integrity following exertion. This
conversion also involves changes in the expression of specific-fiber genes such as
troponin I (slow) and myoglobin genes. Using culture cells, the same group has shown
that this regulation is mediated by PGC-1α co-activation of MEF2 which is a essential
muscle transcription factors involved in fiber type determination [30, 59].Similarly to
PGC-1α , transgenic expression of PGC-1β , causes a marked induction of IIX fibers,
which are oxidative but have "fast-twitch" biophysical properties. PGC-1β also co-
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activates MEF2 transcription factor to bind to type IIX myosin heavy chain (MHC)
promoter and activate its expression. PGC-1β transgenic muscle fibers are rich in
mitochondria and are highly oxidative, at least in part, due to co-activation by PGC-1 β
of ERRα and PPARα . Consequently, these transgenic animals can run for longer and at
higher workloads than wild-type animals [41]. Moreover, in vivo skeletal muscle
experiments have demonstrated that PGC-1α powerfully regulates VEGF (vascular-
endothelial-grow-factor) expression resulting in muscle angiogenesis augmentation.
PGC-1α null mice submit to ischemic insult failure to reconstitute blood flow in a
normal manner to the limb whereas transgenic expression of PGC-1α in skeletal muscle
is protective. Instead, PGC-1α co-activates ERR-α on conserved binding sites found in
the promoter VEGF gene. Thus, PGC-1α and ERR-α also control a novel angiogenic
pathway that delivers needed oxygen and substrates [76].
FIGURE 3: The absence of dPGC-1 leads to muscle fiber degeneration, mitochondrial morphology
and mitochondrial content in null dPGC-1 flies. Transmission electron microscopic analysis of thorax
sections from wt and null dPGC-1 flies are shown. A and B panels illustrate longitudinal sections and C
and D panels show transversal sections. A and C panels represent wild type flight muscle sections and B
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and D panels illustrate altered morphology and mitochondria alignment along the fibers in flight muscle
sections in dPGC -/- flies (arrows and asterisk). (Scale bars=1μm). M, mitochondria; Z, Z axis.
In the other hand, PGC-1β -deficient mice shows milder stress-induced phenotypes but
very similar to PGC-1α null mice [77, 78]. To complete this analysis, double deficient
PGC-1α and PGC-1β null mice were done and the analysis of the phenotype revealed
that they die shortly after birth as result of heart failure, including abnormal cardiac
maturation and block in mitochondrial biogenesis. These studies clearly demonstrate
that both proteins have overlapped targets and therefore, the absence of one of the
members may be partially compensated by the other factor but the absence of both
generates a strong phenotype.
Similar to PGC-1α deficiency mice, Drosophila dPGC-1 null flies are viable and
females are sterile. In addition, adult null flies showed locomotor alterations that
partially unable them to flight (unpublished results). The flight was reduced to 50%,
indicating an important malfunctioning of flight muscles. Muscle and mitochondria
morphologies in null flight muscles were examined to determine whether structural
abnormalities underlay the defects in muscle function. As shown in figure 3, the ultra-
structure of the flight muscles from wild type flies is extremely regular. The myofibrils
are circular in cross section and exhibit regular sarcomeric units that are aligned in
parallel between adjacent myofibrils in longitudinal section. Between these myofibrils a
highly packaged number of electron-dense mitochondria that exhibit the classical
double membrane structure with cristae visualized as invaginations of inner membrane
that extend into the matrix are observed together with a small number of nuclei. In
contrast, flight muscles of mutant null flies exhibit important abnormalities. Thus, most
of the muscles were severely disrupted and a high percentage of muscle fibers were
observed wavy, misalignment and degenerated. Myofibril diameters were variable and
mostly decreased and irregulars. An important increase of connective tissue was
visualized between muscle fibers, together with a modestly reduced number and size of
mitochondria relative to wt muscle control. In addition to being less abundant with
irregular shapes, these organelles exhibit slightly reduced electron density and, instead
of recognizable cristae, contain a granular internal structure. Swollen sarcoplasmic
reticulum-like structures were frequently observed. This muscle phenotype varies in
penetrance, some muscles were only moderately disrupted whereas other muscles in the
same fly are severely affected. Thus, the diminished expression of dPGC-1 protein
17
generates clear muscle abnormalities. These include an increase of connective tissue,
changes in the mitochondria size and rearrangements in mitochondria and nuclei in
Drosophila flight muscles. To corroborate these data COX activity assays and NADH
assays were used to determine mitochondrial mass or OXPHOS activity. Related to fly
muscle changes, recently, Tiefenbock and others, [8] observed a diminution of adult
weight in a hypomorphic allele mutants. Additionally, genes involved in the formation,
maintaining and function of the fiber, including MEF2 and TnI genes, are also reduced
in these null mutants (unpublished results). These observations supplies locomotor
phenotype of dPGC-1 null mutant and also are in agreement with the role of PGC-1 co-
activators family in muscle adaptability function in vertebrates.
Regarding other functions of PGC-1α as modulator of other metabolic events, it also
activates gene regulatory programs involved in hepatic gluconeogenesis [79-81], muscle
glucose uptake [82, 83], adaptive thermogenesis and heme biosynthesis [48, 84]. Recent
studies show that PGC-1α and PGC-1β control mitochondrial capacity in an additive
and independent manner in neurons. These evidences demonstrate that activation or
over-expression of members of the PGC-1 family could be used to compensate for
neuronal mitochondrial loss [85].
The role of the third member of the family, PRC (PGC-1 related co-activator), has been
investigated by stably silencing PRC expression with two different shRNAs. Complete
PRC silencing resulted in a severe inhibition of respiratory energy production associated
with the reduced expression and assembly of respiratory complex. In addition to the
respiratory defect, PRC silencing was also implicated in the inhibition of cell cycle
progression. Global changes in gene expression were consistent with both mitochondrial
and growth phenotypes[86-88]. Recent studies of PRC-loss- function support a role for
PRC in the integration of pathways directing mitochondrial respiratory function and cell
growth [89].
CONCLUDING REMARKS
In this review we have exposed the important role of the PGC-1 co-activator family as
metabolic sensors. These proteins are central players in the control of organ-specific
responses to a wide variety of physiological stimuli as well as different kinds of stress.
We have shown that PGC-1α is, both in mammals and flies, one of the key regulatory
18
factors in tissues with a high energetic demand. Phenotypic adaptations to different
signaling pathways, including physical activity, metabolic demands, neuronal
innervations or ischemia converge on PGC-1 α regulation through its interaction with
diverse transcription factors. These interactions increase mitochondrial biogenesis and
function leading to a higher ATP production through both, OXPHOS metabolism and
fatty acid β -oxidation. PGC-1α has a particularly relevant role in muscle development
and adaptation where this co-factor regulates the expression of fiber specific and NMJ
post-synaptic genes. On the other hand, in response to ischemia, PGC-1α regulates
angiogenesis in muscle, accelerating the recovery of blood flow. In consequence, the
therapeutic potential of PGC-1α against diseases associated with deregulated muscle
function is broad. In fact, PGC-1α has shown an anti-atrophic effect in different
experimental systems.
ACKNOWLEDGMENTS
This research was supported by grants BFU2007-61711BMC from the DGICYT
(Spanish Ministry of Education, Culture and Sports) and S2006-GEN0269, MITOLAB
network, from the Comunidad de Madrid, Spain. We thank Dr Lucia Guerrero for
critical reading of the manuscript and Dr Mark Sefton from BiomedRed for checking
the English usage in this manuscript. Dr. Mª Cruz Marin, postdoctoral researcher, is
supported by MITOLAB network from the Comunidad de Madrid, Spain.
19
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