Aldosterone’s Mechanism of Action: Genomic and Nongenomic Signaling

2.1

Nuclear Mineralocorticoid Receptor and Cytosolic Mineralocorticoid Receptor

The MR (encoded by the NR3C2 gene) belongs to the steroid receptor superfamily that include the progesterone, estrogen, androgen, and the glucocorticoid receptors (GRs). The MR is the longest member of the superfamily of ligand-regulated transcription factors that includes steroid and thyroid hormone receptors, and also the retinoic acid, vitamin D, peroxisome proliferator-activated, and retinoid X receptors.

The structure of MR consists of an N-terminal A/B domain (NTD), responsible for cofactor, the C-domain where DNA binding takes places, with a high homology to the GR, and after a short hinge region comes the C-terminal ligand-binding domain (LBD). The NTD regulates transactivation (mainly by region activation function 1, AF-1) but also interacts with the LBD in an N–C interaction that stabilizes the receptor conformation. The DNA-binding domain (DBD) binds to the hormone response element (HRE) of MR-regulated genes to mediate transcription. The MR LBD is very conserved between species and has multiple functions, that includes, besides ligand binding, also nuclear localization, dimerization, interaction with chaperones, and modulation of transcriptional coactivators and ligand-dependent transactivation.

Interestingly, while aldosterone is known to be the primary physiological MR ligand in humans, in some tissues with scarce 11β-hydroxysteroid dehydrogenase (11β-HSD2) it is believed that cortisol may act as the primary ligand for MR, whereas progesterone behaves as a predominant antagonist.

Classically, all steroid hormone receptors are ligand-activated nuclear transcription factors. In the case of MR (but also for GR and the androgen receptor), in the absence of ligand most MR is located primarily in the cytoplasm. If activated by the proper ligand, MR is shuttled to the nucleus and then back to the cytoplasm after unbound or when transcriptionally inactive.

In its unliganded state in the cytosol, MR is associated with a large heterocomplex of chaperone molecules such as HSP90, HSP70, and p23 that are key players in trafficking but also facilitate the posttranslational modification of the receptors, such as phosphorylation. Recent evidence has shown that the existence of this chaperone complex helps the MR to improve its affinity for ligands and support a dynamic equilibrium between cytosolic and nuclear localization, which can be shuttled in both directions, depending on the presence or absence of ligands. After binding of ligand in the cytosol, MR localization is then shifted to the nucleus by two different modes of MR trafficking. A rapid shifting ( t _1/2 4–10 min) that is mainly regulated by HSP90, as confirmed by using an HSP90 inhibitor such as geldanamycin. However, a slower transport to the nucleus ( t _1/2 40–60 min) has now been described as well. Interestingly, accumulation of MR in the nucleus is still possible in the presence of geldanamycin mediated, in part, by a slower transport mechanism that may be reflective of MR diffusion.

Other associated proteins in the cytosol are involved in MR transport such as dynein/dynactin and the immunophilin FKBP52. To favor the cytoplasmic transport of MR to the nucleus, FKBP52 links the MR–HSP90 complex to dynein/dynactin motors, thus improving nucleocytoplasmic trafficking. Next, the MR trafficking to the nucleus is possible through an active transport that involves nuclear pore complexes and the binding of MR to importin α. Finally in the nucleus, MR signaling by homodimerization occurs after dissociation from chaperone HSP90. Of interest, it has also been postulated that MR is capable of forming heterodimers with other steroid receptors, in particular the GR and ER, which offers additional transcriptional regulation.

2.2

Cell Membrane Mineralocorticoid Receptor

Currently, a large body of evidence supports the view that aldosterone effects are mediated via the classic cytosolic MR protein in various cell types but at least a small fraction of MR is also located in the membrane.

In analogy to the estrogen receptor (ER), it was hypothesized that MR localized to the membrane and could be responsible for different activation pathways than those mediated via MR’s nongenomic or rapid aldosterone effects. For example, using coimmunoprecipitation and fluorescence imaging techniques, it was demonstrated that MR colocalized with epidermal growth factor receptor (EGFR), a tyrosine kinase receptor at the plasma membrane, inducing extracellular signal–regulated kinase (ERK) phosphorylation. In addition, other downstream signaling molecules potentially modulated by MR–EGFR cross talk include NADPH oxidase, the proto-oncogene c-Src, protein kinase C, calcium, reactive oxygen species, and small GTPase.

In the cell membrane, small invaginations called caveolae are not only a structural platform for receptors and enzymes but also act as functional modulators for different cellular pathways. The main component of plasma membrane caveolae, caveolin 1 (cav-1), has an important role in signal transduction and intracellular trafficking and interacts with several steroid receptors, such as the ER. Interestingly, Pojoga and colleagues have shown that cav-1 colocalizes and coimmunoprecipitates with the MR in renal and cardiac tissues and that MR–cav-1 complexes can be modulated and are more abundant during Na ⁺ loading ( Fig. 1 ).

Also, since some studies have found that some of the rapid aldosterone-mediated effects are not blocked by MR antagonism, identifying an alternate aldosterone receptor in the membrane is an ongoing area of research. Likewise, Gros et al. have proposed that MR-independent effects of aldosterone are mediated by the G protein-coupled estrogen receptor (GPER). Interestingly, GPER expression is required for rapid MR-independent effects of aldosterone in VSMC, and can be abolished by a GPER antagonist, decreasing ERK½ phosphorylation. Also the cross talk of MR with the angiotensin II (AngII) receptor type 1 (AT ₁ R) and the vascular endothelial growth factor (VEGF) receptor are under current research.

2.3

Coregulators of Aldosterone Signaling

Over the past decade, we have learned that rapidly activated signaling cascades that can act as coactivators and corepressors modulate aldosterone-induced transcription. These heterogeneous groups of coregulators may enhance or repress nuclear receptor-mediated transactivation of target genes and are currently crucial to our understanding of the complexity of MR signaling, especially in relation to ligand- and tissue-specific activation.

Coactivators are usually large complexes associated with target genes that perform or regulate enzymatic reactions needed for gene expression such as initiation of transcription, histone modification, or RNA splicing.

Steroid receptor coactivator 1 (SRC-1) was the first nuclear receptor coregulator recognized. SRC-1 is a coactivator that binds to the activation function 2 (AF-2) region in the MR LBD and recruits histone acetylation complex to initiate transcription. Other MR coactivators described are the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) interacting with AF-2 in LBD and the transcription-intermediary factor (TIF-1) binding to the NTD region. On the other hand, several corepressors have been described regulating histone activity, apoptosis, and repressing transactivation. For example, both nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptor (SMRT), decrease ligand-dependent MR transactivation by inducing histone deacetylase activity via LBD interaction.

Although many other proteins have been identified as potential coregulators by in vitro screening, additional data is needed, especially related to the clinical relevance for new and more specific MR antagonists.

2.4

Mineralocorticoid Receptor Interaction With the Estrogen Receptor

There is growing evidence of a functional and physical interaction of MR and the ER as mentioned above. Lisanti’s group showed evidence that cav-1 acts as a positive modulator of estrogen signaling and that cav-1 interacts with ER-α in cotransfected 293T cells. Caveolins are principal molecules within the caveolae that compartmentalize various cellular functions in the plasma membrane. In ECs, Chambliss et al. reported that ER-α localized to the caveolae where it coupled with the endothelial nitric oxide (NO) synthase (eNOS) and its activity. Karas’ group then identified striatin, a cav-1-binding protein, that anchored the ER-α, eNOS, and the Gαi complex to the membrane to mediate the rapid nongenomic effects of estradiol on eNOS activation. Of interest, Pojoga et al. showed coimmunoprecipitation studies suggesting the presence of a cav-1/MR interaction in vascular tissue that was most likely mediated via the evolutionary conserved cav-1-binding motif within the N-terminal region of MR. In addition, they showed that cav-1 mediated the rapid/nongenomic effects of aldosterone on ERK½ phosphorylation in human ECs. Coutinho et al. expanded on these findings and showed that cav-1 and MR formed a complex with striatin in human and mouse vascular tissue. They showed that striatin mediated aldosterone’s rapid, nongenomic effects and that aldosterone preincubation could enhance estrogen’s rapid, nongenomic effect on eNOS activation (phosphorylation) in ECs in part by increasing striatin levels. This effect was specific for aldosterone as knockdown of striatin in vascular cells had no significant effect on epidermal growth factor activation. Thus MR, ER-α, striatin, and cav-1 form a complex of receptors in the membrane that may also include the androgen receptor among others. However, the interaction of MR and ER-α is not entirely clear and additional in vivo and in vitro studies in male and female animal models and humans are critically needed in this area. To this end, Barrett Mueller and colleagues showed that MR and ER-α can coimmunoprecipitate from HEK293 cells that were cotransfected with these receptors. However, striatin failed to be detected in this complex. Nonetheless, they reported that ER-α can block MR-mediated genomic activation and demonstrated that estradiol treatment prevented aldosterone-stimulated ICAM-1 increases and leukocyte adhesion in EC lines that express ER-α.

2.5

Mineralocorticoid Receptor Ligands and Their Selectivity

From an evolutionary perspective, to survive the transition from aquatic life to a land environment with limited salt availability and day–night cycles, new complex mechanisms have been developed for Na ⁺ retention and circadian regulation. Phylogenetic analyses showed that adrenocortical hormone secretion and steroid receptors coevolved during the different stages of vertebrate evolution, possibly to improve receptor selectivity and helping diversification and adaption to new environments. This novel information sheds further insight on many mysteries of adrenal physiology, such as prereceptor regulation and the versatility of MR that can be activated by ligands other than aldosterone, such as cortisol or deoxycorticosterone. Furthermore, these phylogenetic studies have demonstrated that genes encoding the machinery for cortisol secretion preceded those for aldosterone secretion. Since cortisol, which has high MR affinity, is more abundant than aldosterone, 11β-HSD2 evolved as a gatekeeper for inappropriate MR activation by cortisol, thus improving tissue selectivity, especially in the kidney. This prereceptor regulation is crucial for modulating receptor activation and ligand specificity. On the other hand, the close homology of the MR and GR is explained by a shared ancestral corticoid receptor (CR). Despite a similar structure at a receptor level, mineralocorticoid selectivity is also achieved by ligand-induced conformational changes, as aldosterone dissociates more slowly from the MR and induces greater transactivation than cortisol at any given concentration. Finally, at the postreceptor level, several coregulators have been recognized to play a key role in tissue specificity and ligand specificity, endorsing aldosterone-, rather than cortisol-mediated, activation of the MR.