ReviewVolume 5 | 2007
Cite this Article: Nuclear Receptor Signaling (2007) 5, e012.
The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology
Inserm U693, Le Kremlin-Bicêtre, France and Univ Paris-Sud, Faculté de Médecine Paris-Sud, UMR-S693, Le Kremlin Bicêtre, France
The last decade has witnessed tremendous progress in the understanding of the mineralocorticoid receptor (MR), its molecular mechanism of action, and its implications for physiology and pathophysiology. After the initial cloning of MR, and identification of its gene structure and promoters, it now appears as a major actor in protein-protein interaction networks. The role of transcriptional coregulators and the determinants of mineralocorticoid selectivity have been elucidated. Targeted oncogenesis and transgenic mouse models have identified unexpected sites of MR expression and novel roles for MR in non-epithelial tissues. These experimental approaches have contributed to the generation of new cell lines for the characterization of aldosterone signaling pathways, and have also facilitated a better understanding of MR physiology in the heart, vasculature, brain and adipose tissues. This review describes the structure, molecular mechanism of action and transcriptional regulation mediated by MR, emphasizing the most recent developments at the cellular and molecular level. Finally, through insights obtained from mouse models and human disease, its role in physiology and pathophysiology will be reviewed. Future investigations of MR biology should lead to new therapeutic strategies, modulating cell-specific actions in the management of cardiovascular disease, neuroprotection, mineralocorticoid resistance, and metabolic disorders.
11β-HSD2: 11β-hydroxysteroid dehydrogenase 2; ACE: angiotensin converting enzyme; ACTH: adrenocorticotrophic hormone; ADAMTS1: a disintegrin and metalloproteinase with thrombospondin-like motifs 1; adPHA1: autosomal dominant pseudohypoaldosteronism type 1; AF1: activation function 1; AF2: activation function 2; ANF: atrial natriuretic factor; AR: androgen receptor; ASC2: activating signal cointegrator 2; BMP2: bone morphogenetic protein 2; CBP: CREB binding protein; CHIF: channel-inducing factor; CNS: central nervous system; DAXX: death-associated protein 6; DBD: DNA binding domain; EGF-R: epidermal growth factor receptor; Egr-1: early growth response gene-1; ELL: eleven-nineteen lysine-rich leukemia; ENaC: epithelial sodium channel; ERK: extracellular signal-regulated kinase; ET-1: endothelin-1; FAF1: Fas associated factor 1; FLASH: FLICE associated huge; G6PD: glucose-6-phosphate dehydrogenase; GILZ: glucocorticoid-induced leucine zipper protein; GR: glucocorticoid receptor; GRE: glucocorticoid responsive element; HAS2: hyaluronic acid synthase 2; HDAC: histone deacetylase; hMR: human mineralocorticoid receptor; HRE: hormone responsive element; hsp: heat shock protein; KS-WNK1: kidney specific with no lysine [K] kinase 1; LBD: ligand binding domain; LXRβ: liver X receptor β; MAPK: mitogen-activated protein kinase; MDM2: murine double minute gene 2; MR: mineralocorticoid receptor; MRE: mineralocorticoid responsive element; NAD: nicotinamide adenine dinucleotide; NCoR: nuclear receptor corepressor; NDRG2: N-Myc downstream regulated gene 2; Nedd: neuronal precursor cell-expressed, developmentally down-regulated gene; NES: nuclear export signal; NLS: nuclear localization signal; NR: nuclear receptor; NTD: N-terminal domain; Orm: orosomucoid; p/CAF: p300/CBP-associated protein; PAI-1: plasminogen activator inhibitor-1; PDK1: 3-phosphoinositide-dependent kinase 1; PGC-1α: peroxisome proliferator-activated receptors γ (PPARgamma) coactivator-1 α; PGC-1β: PPAR γ coactivator-1 β; PIAS: protein inhibitor of activated signal transducer and activator of transcription; PR: progesterone receptor; RGS2: regulator of G-protein signaling 2; RHA: RNA Helicase A; RIP140: receptor-interacting protein 140; RXRβ: retinoid X receptor β; SGK1: serum and glucocorticoid-regulated kinase 1; SMRT: silencing mediator of retinoid and thyroid hormone receptors; SR: steroid receptor; SRC-1: steroid receptor coactivator-1; STAT: signal transducer and activator of transcription; SUMO: small ubiquitin-like modifier; TIF: transcription-intermediary-factor; TM: transcriptional machinery; TNX: Tenascin-X; Ubc9: ubiquitin-like protein SUMO-1 E2-conjugating enzyme 9; UCP-1: uncoupling protein-1; UPAR: urokinase-type plasminogen activator receptor; Usp2-45: ubiquitin-specific protease 2-45
In the late 1960s, evidence for the presence of specific receptors mediating corticosteroid action in the toad bladder was initially proposed by the group of Edelman [Porter and Edelman, 1964]. Subsequently, Type I and Type II corticosteroid receptors were described and identified as mineralocorticoid (MR) and glucocorticoid receptors (GR) [Marver et al., 1974]. MR was characterized as a high affinity (Kd~1 nM), low capacity (20-50 fmol/mg protein) receptor and demonstrated to be a major regulator of sodium reabsorption in the kidney [Funder et al., 1972]. Fifteen years later, the human MR (hMR) cDNA was cloned by the Evans laboratory by screening a human kidney cDNA library at low stringency with a probe encompassing the DNA binding domain of the GR [Arriza et al., 1987]. MR was subsequently cloned and characterized in many species including Xenopus, fish (zebra fish, teleost fish [Greenwood et al., 2003], rainbow trout [Sturm et al., 2005]), bird [Hodgson et al., 2007; Porter et al., 2007] and mammals (mouse, rat [Patel et al., 1989], mole, pig, cow, monkey [Patel et al., 2000; Pryce et al., 2005]). In the late 1990s came the identification of multiple transcription coregulators that mediate MR transcriptional potency at aldosterone-target genes, as reviewed by [O'Malley, 2007]. MR is now recognized as a crucial transcription factor involved in many physiological processes and pathological disorders.
The gene NR3C2 encoding the hMR is located on chromosome 4 in the q31.1 region and spans approximately 450 kb [Morrison et al., 1990; Zennaro et al., 1995]. As illustrated in Figure 1, the gene is composed of ten exons; the first two exons, 1α and 1β, are untranslated, and the following eight exons encode the entire MR protein of 984 amino acids (aa). The rat MR gene is located on chromosome 19q11 and differs slightly in having three untranslated exons (1α, 1β and 1γ) and encoding a 981 aa protein [Kwak et al., 1993]; a similar genomic structure is found for mouse MR gene, which encodes a 978 aa protein. In addition, it now appears that the MR gene does not encode only one protein, but gives rise to multiple mRNA isoforms and protein variants [Pascual-Le Tallec and Lombes, 2005], thus allowing combinatorial patterns of receptor expression potentially responsible for distinct cellular and physiological responses in a tissue-specific manner.
Like all members of the nuclear receptor superfamily, MR has three major functional domains; a N-terminal domain (NTD), followed by a central DNA-binding domain (DBD), and a hinge region linking them to a C-terminal ligand-binding domain (LBD). Exon 2 encodes most of the NTD, small exons 3 and 4 for each of the two zinc fingers of the DBD, and the last five exons for the LBD (Figure 1).
The MR NTD is the longest among all the steroid receptors (SR), (602 aa). The NTD is highly variable among SR, showing less than 15% identity, but for a given receptor, highly conserved between species (more than 50% homology), strongly suggesting a crucial functional importance. The NTD possesses several functional domains responsible for ligand-independent transactivation or transrepression, as shown schematically in Figure 1. Two distinct activation function 1 domains (AF1), referred to as AF1a (residues 1-167) and AF1b (residues 445-602), have been demonstrated in both rat [Fuse et al., 2000] and human MR [Pascual-Le Tallec et al., 2003]. A central inhibitory domain (residues 163-437) has also been characterized and seems to be sufficient to attenuate the overall transactivation strength of the NTD fused either to AF-1a or AF-1b [Pascual-Le Tallec et al., 2003]. These different domains of the NTD recruit various coregulators responsible for modulating the transcriptional activity of MR in a highly selective manner compared with other SR, and are now considered to be important determinants of mineralocorticoid selectivity [Pascual-Le Tallec and Lombes, 2005].
The DBD has the ability to recognize specific target DNA sequences or hormone response elements (HRE). The MR DBD is 94% identical with that of GR, and more than 90% compared with the progesterone receptor (PR) and the androgen receptor (AR). The MR DBD is a 66 aa domain encoded by exons 3 and 4: by analogy with the crystal structure obtained of the GR DBD, it contains two perpendicular α helices, structurally coordinated by a zinc ion that interacts with four cysteine residues and is thus responsible for the zinc finger’s structure. The first zinc finger contains the “P box” (defined by the three residues Gly621Ser-Val625) responsible for tight binding to the minor groove of the DNA double helix. The second zinc finger facilitates receptor dimerization through the so-called “D box” (Ala640GlyArgAsnAsp645), located in the N-terminal part of the DBD. In this context, it is interesting to note that MR is able to heterodimerize with other members of the SR subgroup, most notably GR and AR [Liu et al., 1995], consistent with the possibility that heterodimerization might play a role in some physiological responses at the level of transcriptional regulation.
The MR LBD is a complex and multifunctional domain that spans 251 aa. It is relatively conserved among SR (~55% homology) and highly conserved across species (80-97% homology), and allows selective hormone binding, thus transducing endocrine messages into specific transcriptional responses. The MR LBD crystal structure has recently been solved, thus confirming the remarkable similarity in structure among all SR [Bledsoe et al., 2005; Fagart et al., 2005; Li et al., 2005]. Basically, the MR LBD consists of 11 α helices and four small antiparallel β strands that fold into a three layer helical sandwich. Solving the crystal structure allowed identification of the crucial amino acid residues interacting with the functional group of steroid ligands. For instance, Gln776 of helix H3 and Arg817 at the end of helix 5 directly contact the 3-ketone group of aldosterone, and Asn770 of helix H3 stabilizes aldosterone 18-hydroxyl group. Other residues in helices 6 and 7, as well as Thr945 on helix 10, also directly contact the steroid ligand. A single residue at position 848 in helix H7 switches hormone specificity between MR and GR [Li et al., 2005], and amino acids 820-844 (which are not part of the ligand binding pocket) are also critical for aldosterone binding and ligand binding selectivity [Rogerson et al., 2007]. The role of Met852 in accommodating the C7 substituents of antimineralocorticoid spirolactones has very recently been described [Huyet et al., 2007]. Finally, on the basis of the high similarity between the LBD of MR and GR, and considering the evolutionary tree of this receptor subgroup, it has been proposed that MR was closer to the primordial ancestral corticosteroid receptor [Hu and Funder, 2006], which has been proposed as having high affinity for aldosterone, well before the hormone appeared [Bridgham et al., 2006]. In this context, it is also interesting to note that the Ser949 in human MR is deleted in almost all GR and that the His950 in human MR, conserved in MR in Old World monkeys, is a glutamine in all teleost and land vertebrate MR and may thus represent a breaking point during evolution [Baker et al., 2007].
The MR LBD possesses a ligand-dependent AF-2 constituted by the helices H3, H4, H5 and H12. Upon ligand binding, a rearrangement of the LBD occurs: H12 closes over the ligand pocket which, in combination with the bending of helices H3, H5 and H11, forms a hydrophobic cleft on the surface of the LBD. This groove serves as a docking surface for transcriptional coactivators possessing a NR box defined by the LXXLL motif, an interaction essential for activation of MR transcriptional activity. Given the high level of homology between their LBDs, it is not surprising that GR and MR recruit almost identical coactivators through their AF2 domains. A recent study, using an isolated MR LBD as bait to screen interaction with a LXXLL peptide library showed that MR interacts with a restricted number of coactivator peptides including SRC-1, ASC2, PGC-1α and PGC-1β [Hultman et al., 2005].
Although the relative contributions of AF1a, AF1b and the AF2 to MR transcriptional activity appears to be highly dependent on cellular and promoter contexts, the NTD appears to account for ~40-50% of total transactivation and represents a key determinant of MR specificity [Pascual-Le Tallec and Lombes, 2005].
As noted above, MR is expressed as at least two different proteins, MRA and MRB [Pascual-Le Tallec et al., 2004], resulting from strong Kozak sequences initiating alternative translation. These variants display distinct transactivation capacities in vitro; it remains to be established whether they are differentially expressed in vivo, and the extent to which they contribute to fine-tuning of MR transcriptional activity and the differential patterns of gene expression in different cellular contexts, as previously described for GR [Lu and Cidlowski, 2005].
In its non-liganded state, MR interacts with a large variety of proteins, most notably in the cytoplasmic compartment, thus forming part of a hetero-oligomer [Rafestin-Oblin et al., 1989]. MR contacts chaperone proteins such as the heat shock protein hsp90 [Binart et al., 1995], and indirectly interacts with hsp70, the p23 and p48 proteins and the FKBP-59 immunophilins or CYP40 cyclophillin [Bruner et al., 1997; Pratt and Toft, 1997]. These chaperones play a pivotal role in maintaining MR in an appropriate conformation for ligand binding. Besides the chaperone proteins, the MR also interacts with actin [Jalaguier et al., 1996], which may thus play a role in the ligand-dependent nuclear translocation. Upon hormone binding, the MR dissociates from chaperone proteins, undergoes nuclear translocation and interacts with numerous molecular partners in a coordinate and sequential manner to ensure appropriate transcriptional regulation. For over a decade, yeast two-hybrid screening, GST pulldown and coimmunoprecipitation assays have been used to identify various MR-interacting nuclear proteins.
From a functional point of view, the most important are the transcriptional coregulators acting either as coactivators or corepressors of MR transactivation. Since the initial description of coactivators in the mid 1990s, our knowledge of the complexity of transcriptional regulation has considerably increased. SR are now considered as platforms recruiting in an ordered and cyclical manner different coregulators [Metivier et al., 2003], which exhibit various enzymatic activities to play the role of transcriptional master switches [O'Malley, 2007]. The first member of the large coactivator family identified was steroid receptor coactivator-1 (SRC-1) [Onate et al., 1995], postulated to initiate transcription by recruiting a series of proteins involved in chromatin remodeling, histone acetylation and methylation [Freiman and Tjian, 2003; Rosenfeld et al., 2006]. Since then, a dozen coregulators have been demonstrated to interact with MR and modulate its activity (for an exhaustive review on this topic, see [Pascual-Le Tallec and Lombes, 2005]). As summarized and referenced in Table 1, the most important coactivators for MR transactivation appeared to be the histone acetylase CBP/p300; the helicase RHA; the transcriptional coactivators SRC-1, SRC-1e, and PGC-1; and finally, the Pol II elongation factor ELL, that constitutes the first example of a selective transcriptional coregulator of MR [Pascual-Le Tallec et al., 2005]. Corepressors able to bind MR and repress its transcriptional function include the widely repressive SMRT and NCoR; the apoptosis regulator DAXX; and the specific SUMO-ligase PIAS proteins. Of interest, sumoylation now emerges as an important posttranslational modification for many nuclear receptors and coregulators. SUMO-E3 ligase PIAS proteins repress MR, potentially in collaboration with DAXX protein [Lin et al., 2006]; it has recently been shown, however, that the SUMO-E2 activating enzyme Ubc9 interacts with the MR NTD/DBD (1-670 aa) to potentiate aldosterone-dependent MR transactivation [Yokota et al., 2007], further increasing the complexity of sumoylation-mediated regulation. The MR interaction network per se appears not to determine functional MR activity; rather, the coordination and sequential interaction of molecular partners, directly or indirectly with MR, controls activation and function of cooperative transcriptional complexes, probably in a cell- and promoter-dependent manner. It should be mentioned that MR also heterodimerizes with other SR, notably GR and AR [Liu et al., 1995; Savory et al., 2001], thus providing additional support for MR functional diversity of action.
Until the late 1970s, MR expression measured by binding assays was considered restricted to polarized tight epithelia, which show aldosterone-dependent transepithelial sodium transport [Marver et al., 1974]. MR expression was located by immunohistochemistry in the kidney, most notably in the distal convoluted tubules and cortical collecting ducts [Krozowski et al., 1989; Lombes et al., 1990]. MR seems also to be expressed at the messenger and the protein levels in glomeruli, especially in mesangial cells [Miyata et al., 2005; Nishiyama et al., 2005] and podocytes [Shibata et al., 2007], where aldosterone has been reported to modulate podocyte function, possibly through the induction of oxidative stress and of the serum and glucocorticoid-regulated kinase 1 (SGK1). MR expression was also detected, by specific binding of [3H]-aldosterone, in the distal colon of rat [Pressley and Funder, 1975], human [Lombes et al., 1984] and chick [Rafestin-Oblin et al., 1989]. The lung may represent another aldosterone target tissue, in that MR binding sites were demonstrated in airway epithelia from bronchiole to trachea [Krozowski and Funder, 1981]. MR expression (transcript and/or protein) was clearly revealed in the salivary [Funder et al., 1972] and sweat glands [Kenouch et al., 1994], in the liver [Duval and Funder, 1974] and in the inner ear [Furuta et al., 1994; Pitovski et al., 1993; Teixeira et al., 2006]. Importantly, epithelial expression of MR was always associated with expression of the 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), the enzyme that allows aldosterone to selectively activate MR, by converting glucocorticoid hormones to their 11-keto analogs, unable to bind MR [Edwards et al., 1988; Funder et al., 1988].
Subsequently, MR expression was detected in non-epithelial tissues in which the expression of 11β-HSD2 was absent or extremely low. For instance, specific binding sites for aldosterone were identified in mononuclear leucocytes [Armanini et al., 1985] and in the heart [Barnett and Pritchett, 1988; Pearce and Funder, 1987] and MR transcripts were detected in specific structures of the hippocampus (dentate gyrus and CA1, 2, and 3 nuclei) and in the hypothalamus [Han et al., 2005; Herman et al., 1989; Van Eekelen et al., 1988]. In 1992, MR was localized at the cellular level by immunohistochemistry in cardiomyocytes, endothelial cells and large vessels [Lombes et al., 1992]. Some years later, MR expression was confirmed, at the messenger and protein level in the skin, not restricted to sweat and sebaceous glands, but also in keratinocytes constituting the stratified epithelium [Kenouch et al., 1994]. Recent studies have shown MR to be expressed at the transcript and protein level in adipose tissues, both in white [Caprio et al., 2007; Fu et al., 2005; Rondinone et al., 1993] and brown adipocytes [Penfornis et al., 2000; Viengchareun et al., 2001; Zennaro et al., 1998]. This is of particular interest considering the functional interaction between MR and PGC-1 [Hultman et al., 2005] and the central role of this coactivator for brown adipocyte differentiation [Lin et al., 2005]. Of note, MR is also expressed at the protein level in ocular tissues, such as retina [Mirshahi et al., 1997] and iris-ciliary body [Schwartz and Wysocki, 1997], in placenta [Hirasawa et al., 2000], and at the messenger level in uterus, ovaries and testis [Le Menuet et al., 2000], with no clear roles reported to date.
This widespread expression of MR suggests novel functions for this receptor in these target tissues. It also raises questions regarding the role played by glucocorticoid hormones (cortisol or corticosterone) in MR activation, considering the absence of 11β-HSD2 in non-epithelial tissues, except in certain brain areas such as the nucleus of the solitary tract, as recently reported [Geerling et al., 2006; Naray-Fejes-Toth and Fejes-Toth, 2007].
MR is now considered an ubiquitous transcription factor, and real-time PCR quantification of MR and GR transcripts reveals interesting anatomical expression patterns [Bookout et al., 2006]. MR and GR expression are equivalent and high in the gastrointestinal system, and moderate in the endocrine, reproductive, metabolic and cardiovascular systems. MR expression is higher than that of GR in the central nervous system (CNS) and the structural system (skeleton), whereas GR expression is, as expected, more pronounced than that of MR in the immune system. According to the classification based on hierarchical clustering of gene expression, MR seems to belong to the same cluster as LXRβ and RXRβ, the expression of which is most abundant in the CNS and is crucial to the global basal metabolism, linked to circadian clocks, metabolism and cardiovascular control [Bookout et al., 2006]. Thus, distinct MR and GR expression patterns strongly support that MR and GR differentially affect transcriptional programs governing distinct physiological processes and pathophysiological disorders.