Regulation of the expression of the estrogen related receptors (ERRs)

Abstract

Estrogen related receptors (ERRα, β and γ in mammals) are orphan members of the nuclear receptor superfamily acting as transcription factors. ERRs are expressed in several tissues and cells and they display various physiological and pathological functions, controlling, amongst others and depending on the receptor, bone homeostasis, energy metabolism, embryonic stem cell pluripotency, and cancer progression. In contrast to classical nuclear receptors, the activities of the ERRs are not controlled by a natural ligand. Regulation of their activities thus rely on other means such as post-translational modification or availability of transcriptional co-regulators. In addition, regulation of their mere expression under given physiological or pathological conditions is a particularly important level of control. Here we discuss the mechanisms involved in the regulation of ERRs expression and the reported means to impact on it using pharmacological approaches.

Introduction

Nuclear receptors (NRs) are classically defined as transcription factors, whose activities are modulated by a ligand. NR proteins comprise a number of conserved domains among which a centrally located DNA binding region as well as a C-terminally located ligand-binding domain. The latter displays the capacity to regulate transcription, mainly by interacting with transcriptional co-modulators. However, for a number of NRs, no natural ligand has been identified to date. These particular NRs as thus referred to as “orphan” nuclear receptors. Falling within this category are the members of the Estrogen Related Receptor (ERR) subfamily, comprising three isoforms in mammals (ERRα, β and γ), that originate from distinct genes, ESRRA, ESRRB and ESRRG, respectively [1, 2].

ERRs are broadly, but not ubiquitously, expressed during mouse embryonic development and in the adult in a partly overlapping, partly distinct pattern [3] (summarized on Fig. 1). At least a number of their respective physiological and pathological functions have been determined, using various approaches including genetically modified animal models. ERRα and γ are highly involved in energy metabolism, bone homeostasis and cancer progression, amongst others (reviewed in [2–6]). Consistent with its expression pattern, the functions of ERRβ are more restricted, albeit not exclusively, to the maintenance of pluripotency in embryonic stem cells (ESC) in the mouse [7].

Fig. 1.

Summary of generalities on ERR (α, β and γ) receptors. Schematic organization of the ERR receptors is depicted in the middle panel, with the DNA-binding domain (DBD) and the C-terminally located ligand binding domain (LBD) which comprises the transcriptional activation functions. Selected traits of expression pattern of the ERRs are summarized on the left panel. Selected functions of ERRs are summarized on the right panel. ESCs embryonic stem cells

Per definition, the activities of orphan NRs, including ERRs, are not regulated by the presence of a natural ligand, as opposed to those of “classical” NRs (e.g. estrogen or androgen receptors). These activities are thus regulated by other means that have been at least partly identified. Among these, post-translational modifications are instrumental. For instance, phosphorylation, sumoylation and acetylation of the N-terminal domain of ERRα have been shown to modulate the receptor’s activities [8, 9]. An important level of control is also the presence or absence of given transcriptional co-modulators [10]. However, regulation of the mere expression of ERRs is also a strong way to regulate their activity and has been found highly important in some issues. As an example, high expression and activities of ERRα or ERRγ are correlated to a poor or favorable prognosis, respectively, in several cancers [11]. Consequently targeting the ERRs has been proposed as a possible therapeutic approach against cancers. Various regulations of ERRs expression have been documented at the transcriptional, post-transcriptional and post-translational levels that will be described below. In addition, artificial means have also been identified that impact on ERRs expression.

The purpose of this review is to summarize our current knowledge in the regulation of the expression of ERRs as well as to discuss the possible consequences of these regulated expressions in terms of physiological and/or pathological outputs, when documented.

Transcriptional regulation of ERR expression

Regulated expressions of ERR receptors at the promoter/mRNA level (summarized on Fig. 2) have been documented in several publications. Various reports have shown a rhythmic (circadian) expression of all ERR mRNAs in several mouse tissues, with an expression peak that differs from one receptor to another. For instance, in adipose tissues and liver, the expression of ERRα and β peaks at zeitgeber time (ZT) 12–16, whereas that of ERRγ is maximal at ZT8 [12]. Interestingly, the expression peak of a given receptor may also vary according to the tissue (e.g. ERRβ expression is maximal at ZT4 in some parts of the nephron), suggesting different modes of regulation [13]. It is likely that these variations of expression are directly controlled by the circadian clock as, at least for ERRα, they are abolished in Clock^−/− mice [14] and as the Clock and Bmal transcription factors directly bind the ERRα, β and γ promoters [15, 16]. No evidence are available concerning a potential rhythmic expression of the ERR proteins. However, the half-life of 4–6 h for ERRα protein in cell culture [17, 18] is consistent with the hypothesis of a circadian regulation of mRNA expression impacting the protein level. Noteworthy, the ERRs are highly involved into various types of metabolic processes; a circadian regulation of their expression could thus contribute to metabolic efficiency.

Fig. 2.

Summary of the regulation of the expression of ERRs (α, β and γ as indicated) at the transcriptional level. EDCs endocrine disrupting compounds, NP nonylphenol, BPA and S bisphenol A and bisphenol S, 9-cis-RA 9-cis-retinoic acid. See text for details

A positive auto-regulatory loop has been demonstrated for ERRα, that depends on the PGC-1α co-activator and involves a complex steroid hormone response element (sHRE) located close to the ERRα transcriptional start site [19, 20]. This loop may account at least for part of the stimulation of ERRα mRNA/protein expression in macrophages upon LPS stimulation that is required for the attenuation of inflammatory processes [21]. Similarly, thyroid hormone (TH) enhances PGC-1α expression in liver cells, leading to increased ERRα expression through this auto-regulatory loop [22]. Eventually, this mechanism contributes to TH-induced elevated mitochondrial turnover and activity. In addition, it has been shown that GABPA [which dimerizes with GABPB to form the nuclear respiratory factor 2 (NRF2)] contributes to this autoregulatory loop to induce oxidative phosphorylation in muscle cells [23]. The sHRE also recruits the Estrogen Receptor α (ERα) leading to estrogen-dependent stimulation of ERRα expression in human mammary and endometrial cells as well as in mice uterus and heart [24]. Strikingly, ERRα expression can also be modulated by estrogen receptor agonists and antagonists in an ER-independent manner [25]. In this case, it involves the activation of GPER1, a membrane estrogen receptor not related to the ERs, that stimulates a signaling cascade culminating in the recruitment of various transcription factors, such as Sp1, Sp3 and members of the AP1 complex, to a region located close to the sHRE on the ERRα promoter.

ERRγ also undergoes autoregulation [26, 27]. Indeed its expression is enhanced by various stimuli (e.g. ethanol in liver cells or IL6 in chondrocytes), an increase abolished by exposure to an ERRγ inverse agonist. However, it is not known whether this phenomenon involves a direct binding of ERRγ on its own promoter.

Other transcription factors have been demonstrated as directly regulating the ERR promoters. Among these, nuclear factor erythroid 2-related factor (NFE2L2) represses the expression of ERRα in breast cancer cells [28]. In contrast, the ERRα promoter is directly stimulated by STAT3 in triple negative breast cancer cells, resulting in increased invasion capacities [29]. In prostate cancer cells, the expression of ERRα mRNA and protein is directly stimulated by ERG [30]. Interestingly the ERRα protein in turn increases the expression of the TMPRSS2:ERG fusion gene that is frequently detected in prostate cancers and plays a key role in cancer progression. The ERG-ERRα reciprocal loop may thus contribute to increase prostate cancer aggressiveness. Nanog and TFCP2L1 directly activate the expression of ERRβ in mouse ESC, at least partly accounting for their capacity to maintain pluripotency [31, 32]. The androgen receptor represses ERRγ expression in prostate cancer cells leading to metabolic reprograming [33].

Variations of the level of ERRα and γ through undocumented mechanisms have been observed along some differentiation processes. For instance, the expression of both receptors increases during osteoblast differentiation, a phenomenon that ERRα and γ contribute to limit [34, 35]. In contrast, these receptors promote the in vitro conversion of myoblasts into myotubes (a critical step of muscle cell differentiation), during which their expression is also enhanced [36]. In mature muscles, expression of ERRα and γ is also increased by exercise, leading to enhanced mitochondria-driven oxidative remodeling and muscle fitness [37, 38]. Manipulation of the expression of ERRα and γ may thus result in different outcomes in terms of mature functional cells, depending on the tissue considered.

Exogenous modulation of ERR mRNA

In Trichoplax adhaerens (Ta), a placozoan considered as related to an ancestor of both Cnidarians and Bilaterians, expression of the unique ERR ortholog is stimulated by exposure to nanomolar concentrations of 9-cis-retinoic acid (9-cis-RA) [39]. This compound acts as a ligand for the unique Ta retinoic X receptor (RXR) ortholog. To date, it is unclear: (1) whether 9-cis-RA is a natural ligand of TaRXR, (2) whether TaRXR is required for TaERR stimulation and directly acts on the TaERR promoter, (3) to what extent this regulation is conserved across the animal kingdom. However, these data suggest the existence of a network involving nuclear receptors that could result in metabolic regulations.

An impact of endocrine disrupting compounds (EDCs) on the expression of ERR mRNA has been documented in invertebrates as well as in human. For instance, ethylparaben, a compound widely used in cosmetic and food preservatives, modulates the expression of the unique ERR mRNA in D. melanogaster in a time and dose-dependent manner, although with non-monotonous response curves [40]. Intriguingly, these effects are more pronounced in females than in males. 4-nonylphenol (NP) and di(2-ethylhexyl) phthalate (DEHP) are used in the production of insecticides and plastic fabrication, respectively, and interfere with the steroid (estrogenic and/or androgenic) pathways. Exposure to these compounds up-regulates the expression of the unique ERR mRNA in the fourth instar larvae of Chironomus riparius (a non-biting midge) in a time- and dose-dependent manner [41]. Bisphenol A (BPA) and S were also reported as increasing ERR expression in the same animal [41, 42] as well as in the freshwater snail Physa acuta [43]. Interestingly, a correlation between BPA concentration in urine and the mRNA expression of ERRα, but not β or γ, in blood lymphocytes was documented in human [44]. In this line, it has been shown that nanomolar ranges of BPA (i.e. relevant to general human exposure) increase the expression of ERRα in the Jurkat (T cells) cell line [45]. It should however be pointed out that these EDCs studies focus on mRNA expression without addressing a possible effect on the corresponding promoters. In addition, variations of ERR expression in response to these compounds have not yet been shown at the protein level. EDCs generally affect metabolism and fecundity. However, the consequences of their impact on ERR expression in terms of pathological responses are not documented to date.

Post-transcriptional regulation of ERR expression

The expression of ERRs can also be regulated at different post-transcriptional levels (summarized on Fig. 3). In particular, reports have documented an effect of several microRNAs (miR) on ERRα mRNA. For instance, miR-125a down-regulates the endogenous ERRα mRNA level in porcine preadipocytes via a sequence in its 3′UTR that is conserved in several mammals including human [46]. Since miR-125a decreases preadipocyte differentiation [46] whereas ERRα promotes it [5], it is tempting to speculate that the effect of miR-125a on this process requires ERRα downregulation.

Fig. 3.

Summary of the regulation of the expression of ERRs (α, β and γ as indicated) at the post-transcriptional and post-translational levels. PTMs post-translational modifications. See text for details

Other microRNAs (miR-137, miR-497 and miR-135a) have been shown to decrease the steady state level of ERRα mRNA in breast cancer cells by targeting conserved sequences in the 3′UTR [47–49]. These miRs also inhibit various traits related to cancer progression, such as proliferation, migration and extracellular matrix invasion. The role of ERRα repression in these phenotypes has been shown in the case of miR-137 and miR-135a. Indeed, upon overexpression of these miRs, reintroduction of an ERRα mRNA that displays mutations in the 3′UTR recognition sequences (i.e. escaping the effect of the miRs) rescues the phenotypical defects.

miR-320a and miR-204-5p have been identified as directly targeting ERRγ [50–52].It is possible that these repressive effects impact on parameters of cancer aggressiveness (miR-320a [50, 51]) or myocyte differentiation (miR-204-5p [52]). To the best of our knowledge, no microRNA controlling the expression of ERRβ has been identified to date.

Post-translational regulation of ERR expression

Modulation of ERR protein stability by ubiquitination and proteasome-dependent degradation also appears as an important level in the regulation of expression. For instance, Parkin binds to and ubiquitinates all ERR (α, β and γ) proteins in dopaminergic neurons and also induces ERRα degradation in endothelial cells [53, 54]. In neurons, this leads to a decrease in the expression of monoamine oxidases (MAO) A and B, which are direct target genes of the ERR receptors [55]. MAO enzymes catalyze the oxidation of dopamine, which generates reactive oxygen species and eventually results in neuronal toxicity. The Parkin-mediated ERR degradation thus appears neuroprotective. Strikingly, the mutated Parkin version found in Parkinson Disease (PD) is unable to induce ERR protein degradation, altogether suggesting a link between ERRs and PD.

In mouse liver cells, mTOR (mammalian Target Of Rapamycin) regulates the TCA cycle and lipid biosynthesis. In addition, mTOR controls ERRα ubiquitination and degradation by repressing the expression of the E3 ligase CHIP/Stub1 [17]. The fact that ERRα is also involved in the regulation of the TCA cycle and lipid biosynthesis suggests that, at least part of the effects of mTOR on these metabolic pathways relies on CHIP/Stub1-mediated ERRα degradation. A positive effect of the mTOR pathway on ERRα stability has also been documented in breast cancer cells [56]. Indeed, lapatinib (an inhibitor of receptor tyrosine kinases [RTK]) induces ERRα degradation in drug-sensitive cells. This down-regulation reduces the metabolic adaptations that promote survival of cancer cells. Strikingly, in lapatinib-resistant cells, this degradation is counteracted by the reactivation of the mTOR pathway. Modulation of ERRα expression by RTK- and mTOR pathways thus appears crucial for cancer survival.

O-GlcNAcylation is a process that acts as a nutrient sensor in the liver and maintains energy homeostasis. Upon O-GlcNAcylation, ERRγ ubiquitination is reduced, leading to stabilization of the receptor and to an increased ability to induce gluconeogenesis in vivo [57].

ERRα physically interacts with the histone demethylase LSD1 and switches its activities from a transcriptional repressor to an activator [58]. In turn, LSD1 protects the receptor from ubiquitination and proteasome-dependent degradation, in a manner that does not depend on its enzymatic activity [18]. Although the mechanisms of this protection are unclear, it may contribute to the efficient activation of common ERRα-LSD1 targets that are involved in the induction of cell migration and invasion.

Reports show that ERRα can be phosphorylated through at least two different pathways, a cAMP/PKA dependent one in lung cells [59], as well as an EGF/MEK one in colon cancer cells [60]. These cascades also enhance ERRα protein expression, leading to increased activity at the surfactant protein-A promoter and promoting traits of cancer progression, respectively. The effect of these signaling pathways on ERRα mRNA is unclear, and it is thus tempting to speculate that ERRα phosphorylation results in a stabilization of the protein accounting for the increase of its steady-state level.

Exogenous modulation of ERR protein expression

Although the ERRs are orphan receptors (i.e. again, for which no natural ligand have been identified to date), various synthetic compounds have been isolated that regulate the transcriptional activities of one or several ERR [1, 61]. A majority of these identified compounds act as inverse agonists, reducing the transcriptional activities of the corresponding ERR protein. This phenomenon is generally understood as relying on the disruption of interactions between a given ERR and its cognate transcriptional coactivator(s) [55]. However, some ERRα inverse agonists, such as XCT790, compound A (N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-ylidene]-5H dibenzo[a,d][7] annulen-5-amine), AM251 (originally characterized as a cannabinoid receptor inverse agonist) and C29 have also been shown to destabilize the receptor by inducing its proteasome-dependent degradation [62–65]. Inverse agonists may thus repress ERR activities via two distinct mechanisms, destabilization and disruption of coactivator contacts, although the relative contribution of each phenomenon to the final effect is unclear.

In summary, regulation of the activities of the ERR orphan nuclear receptors is exerted at several levels. Regulation of the mere expression of the receptors appears instrumental in the control of various physiological and pathological processes. In several occurrences, the precise mechanisms through which these regulations are exerted are far from completely understood. However, it is important to point out that manipulation of the expression of the receptors could be an interesting tool to modulate traits associated to given pathological situations.

The existence of synthetic compounds that not only modulate the transcriptional activities of the receptor but also their stability at the protein level provides a proof of concept for this promising pharmacological approach.