Defining the potential role of the mineralocorticoid receptor in musculoskeletal health and bone crosstalk with other tissues

Abstract

Excessive mineralocorticoid receptor (MR) activation in the heart and vasculature leads to pathological effects such as extracellular matrix accumulation, oxidative stress, and sustained inflammation. While MR’s role in cardiovascular and renal systems is well understood, MR signaling has also been implicated as a key driver of homeostasis and pathological changes in several other body systems including skeletal muscle and adipose tissue. The glucocorticoid receptor (GR) and MR are structurally and functionally linked, sharing 95% similarity in DNA binding domains and recognizing many of the same hormone response elements (HRE) as transcriptional regulators of target genes. The role of GR in bone has been defined through several mechanistic studies, whereas the role of MR in bone is understudied. Because mineralocorticoid signaling regulates renal sodium and calcium handling, chronic hyperaldosteronism may indirectly disrupt skeletal homeostasis through urinary calcium wasting and secondary alterations in parathyroid hormone signaling. Furthermore, MR inhibition through MR antagonists (MRAs) has been associated with beneficial skeletal effects, particularly in settings of hyperaldosteronism and 11β-HSD2 deficiency. In this review, we present historical and current scientific findings on the role of genomic MR signaling in bone and extra-skeletal tissues that may be involved in crosstalk with the skeletal system. Furthermore, we also highlight the availability of tools to study MR signaling in the context of the musculoskeletal system.

Introduction:

The mineralocorticoid receptor (MR) is well-known for its role in regulating electrolyte balance in the kidney, sweat glands, salivary glands, colon, and vasculature (1–5). MR belongs to the subclass of nuclear receptors that, upon hormone binding, act as ligand-activated transcription factors (6, 7). Other family members include the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), and glucocorticoid receptor (GR)(8). GR and MR share several similarities in structure and function, with nearly identical amino acid sequences in their ligand binding domain (LBD) and DNA binding domain (DBD); both are able to recognize and bind to DNA sequences known as hormone response elements (HRE; canonical sequence of AGAACAnnnTGTTCT with variations possible) when activated by ligands (9–11) (Figure 1). Despite these structural similarities, GR and MR exhibit distinct transcriptional activities that are influenced by ligand affinity, tissue-specific expression patterns, chromatin accessibility, and coregulator recruitment (12). Ligand-bound MR and GR both have the capability for homodimerization (MR:MR or GR:GR) and heterodimerization (MR:GR or GR:MR) before translocating to the nucleus (7, 10, 13, 14). Chromatin immunoprecipitation (ChIP) assays have demonstrated that MR:GR and GR:MR heterodimers exhibit unique DNA binding specificity and differential gene expression in comparison to their homodimerized counterparts (13). Moreover, these receptor dimerization combinations show functional differences, as certain ligands induce unique conformational changes which influence coregulator interaction and transcriptional activity (10, 15).

Figure 1. Structure of human (h) and murine (m) GR and MR proteins.

Key structural features include the N-terminal domain (NTD), which contains the activating function 1 (AF-1) site, the DNA-binding domain (DBD), and the ligand-binding domain (LBD), which contains the activating function 2 (AF-2) hydrophobic cleft. Figure created with BioRender.com.

Although aldosterone is the classical MR ligand, physiological glucocorticoids such as cortisol (in humans) and corticosterone (in some rodents) bind MR with nearly identical or higher affinity as aldosterone (14, 16, 17). In addition, potent synthetic GR agonists such as dexamethasone or prednisolone also bind to MR but with lower affinity (17). To maintain the specificity of MR activation by aldosterone as compared to glucocorticoids which circulate at 100 to 1000 fold higher concentrations, MR-responsive tissues often demonstrate expression of the intracellular enzyme 11 β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) which catalyzes the conversion of cortisol to cortisone in humans and corticosterone to 11-dehydrocorticosterone (11-DHC) in some rodent species like mice and rats (18, 19). Consequently, 11β-HSD2 acts as a pre-receptor regulatory mechanism that limits glucocorticoid-mediated MR activation despite the substantially higher circulating concentrations of glucocorticoids relative to aldosterone. (18–20).

In many non-epithelial tissues, limited 11β-HSD2 expression permits glucocorticoids to function as the predominant endogenous MR ligands (21, 22). Importantly, even in tissues traditionally considered aldosterone-responsive, cellular heterogeneity in 11β-HSD2 expression creates distinct microenvironments for MR activation (22). In the kidney, brain, and colon, 11β-HSD2-positive cells are often located adjacent to cells that express MR but lack 11β-HSD2 expression. In these 11β-HSD2-negative cells, MR signaling is thought to be driven primarily by glucocorticoids (19, 23). In the kidney, distal nephron principal cells are an example of 11β-HSD2 expressing cells that are adjacent to intercalated cells that lack 11β-HSD2, but express MR (24, 25). The hippocampus is another example where both GR and MR are expressed without the expression of 11β-HSD2 (13, 19, 25). Together, these mechanisms favor MR occupancy by glucocorticoids at basal hormone levels, whereas increasing glucocorticoid concentrations during circadian peaks, stress, or pharmacological exposure favors greater GR activation (19, 26).

In contrast to glucocorticoids, aldosterone circulates at much lower concentrations and exhibits less circadian variation, yet it binds MR with similarly high affinity (27). Consequently, the expression of 11β-HSD2 is crucial for mediating aldosterone specificity thus enabling classical aldosterone-MR signaling (22). Following aldosterone-mediated MR activation, expression and activity of the Epithelial Sodium Channel (ENaC) and Na+/K+-ATPase increase, promoting sodium reabsorption in epithelial tissues such as the kidney and colon (28, 29). Increased sodium influx consequently leads to increased water absorption and reabsorption resulting in increased blood volume and blood pressure. In addition to its epithelial effects, MR activation directly regulates vascular smooth muscle contractility and sympathetic nervous system activity, further contributing to blood pressure regulation (29).

While the role of MR in renal, gastrointestinal, and cardiovascular physiology is well established, its contribution to musculoskeletal tissue homeostasis remains less clearly defined. With this review, we aim to highlight current knowledge gaps, discuss concepts in musculoskeletal MR biology, and summarize the experimental tools available to study MR signaling in musculoskeletal tissues.

Adrenal steroids and bone physiology:

The adrenal cortex consists of three distinct layers: the zona glomerulosa (ZG), which produces mineralocorticoids such as aldosterone, the zona fasciculata (ZF), which produces glucocorticoid, and the zona reticularis (ZR), which produces adrenal androgens (30). Increasing evidence suggests that these adrenal steroids contribute not only to systemic metabolic and electrolyte homeostasis, but also to skeletal physiology and musculoskeletal aging (31, 32). Bone is a dynamic and metabolically active tissue that undergoes constant turnover from the actions of bone-resorbing osteoclasts and bone-forming osteoblasts (33). At present, little is known about the direct impact of ZG-derived mineralocorticoids like aldosterone on bone cells, although clinical literature suggests correlation between high levels of aldosterone and low bone mineral density (BMD), such as occurs in primary aldosteronism (34, 35). The mechanisms underlying these observations remain incompletely understood (36). In addition, findings from human studies have not been entirely consistent; at least one study found that absolute values of aldosterone did not associate with bone parameters derived from peripheral quantitative computed tomography (37), whereas aldosterone levels were negatively correlated with bone mineral density and positively associated with FRAX 10-year fracture probability scores in another study (38). These discrepancies likely reflect differences in patient populations, disease severity, imaging modalities, or study design. It is important to note that circulating aldosterone is tightly regulated by the renin-angiotensin-aldosterone system (RAAS) depending on physiological need (15, 39). Therefore, the aldosterone-to-renin ratio (ARR) is often considered a more reliable indicator of dysregulated aldosterone activity and may provide additional insight into associations between aldosterone excess and skeletal outcomes (37, 40).

ZF-derived glucocorticoids are secreted in a circadian manner and are critical for stress homeostasis, with glucocorticoids peaks occurring during stressful stimuli (30). At physiological levels, glucocorticoids contribute to skeletal homeostasis and osteoblast differentiation (32). In contrast, chronic glucocorticoid excess, such as occurs in Cushing’s syndrome or during prolonged exogenous glucocorticoid therapy, promotes osteoblast and osteocyte apoptosis while increasing osteoclast activity, impairing skeletal integrity (32, 41, 42). The skeletal actions of glucocorticoids are further modulated by receptor expression, chaperone proteins, and local pre-receptor metabolism mediated by 11β-hydroxysteroid dehydrogenase (11β-HSD) enzymes (19). 11β-HSD1 is a nicotinamide adenine dinucleotide phosphate (NADPH) dependent reductase, converting inactive glucocorticoids into active forms, whereas 11β-HSD2 is a nicotinamide adenine dinucleotide (NAD⁺)-dependent dehydrogenase inactivates glucocorticoids (19). Local pre-receptor metabolism, particularly 11β-HSD1-mediated regeneration of active glucocorticoids in osteoblast lineage cells, contributes to glucocorticoid induced trabecular bone loss by suppressing bone formation (43). Together, these local regulatory mechanisms help determine the magnitude and context of glucocorticoid signaling within skeletal tissue.

ZR-derived adrenal androgens, such as dehydroepiandrosterone (DHEA), serve as precursors to sex hormones like testosterone and estrogen. DHEA has been shown to support bone formation and suppress bone resorption either directly or through its conversion into sex steroids (44). Clinically, DHEA supplementation showed some ability to increase bone mineral density (BMD) in aged subjects (60–88 years old) with low baseline levels of DHEA sulfate (DHEAS) (45).

MR signaling biology:

MR is a ligand-activated transcription factor belonging to the steroid receptor family (46, 47). It is involved in diverse physiological functions and is expressed in a range of tissues including the kidney, heart, and brain (48). In the kidney, one of the critical functions of MR is the regulation of electrolyte and fluid balance in the distal nephron (48). Mechanistically, aldosterone binds MR in principal cells of the distal nephron, promoting increased expression and activity of epithelial sodium channels (ENaC) and Na+/K+-ATPase (29, 49). When low blood pressure is detected, aldosterone-mediated MR activation triggers signaling that increases salt and water reabsorption by the kidney, increasing blood volume (49). In addition, MR activation in the paraventricular nucleus (PVN) of the hypothalamus enhances sympathetic outflow (50), and MR signaling in vascular smooth muscle cells exerts rapid non-genomic effects on calcium channels, increasing vascular reactivity to angiotensin II and catecholamines, thereby promoting vasoconstriction, increasing blood pressure (51).

In the absence of ligand binding, MR remains transcriptionally inactive and resides in a cytoplasmic chaperone complex that includes HSP90, HSP70, p23, FK506-binding proteins, and cyclophilins (15). Upon ligand binding, a conformational change forms a hydrophobic cleft known as activation function 2 (AF-2) (Figure 1), which serves as a platform for the binding of transcriptional coregulators (15). The MR then homodimerizes or heterodimerizes with GR and translocates to the nucleus where it binds to HRE in the promoter of MR-regulated genes (Figure 2) (10, 15). As discussed above, ligands for MR include aldosterone and glucocorticoids; cortisol and corticosterone. Progesterone, spironolactone, eplerenone, and finerenone act as MR antagonists, competitively binding to the receptor and inhibiting its activation by endogenous agonists such as aldosterone and deoxycorticosterone (DOC) (8, 25). Several factors determine the outcome of MR modulation. For example, different ligands induce unique conformational changes that influence the binding of various coactivators or corepressors. SRC-1, SRC-2 and PGC-1α are well-known coactivators that potentiate MR effects (15). Meanwhile, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptor (SMRT) are involved in transcriptional repression. In addition, post-translational modification also adds to the complexity of MR regulation (15). MR activation regulates transcription of genes including ENaCα and SGK1 and also modulates signaling pathways such as PI3K/Akt (8, 52).

Figure 2. MR activation by adrenal-derived ligands.

Adrenal-derived ligands, including aldosterone, DOC, or glucocorticoid, are lipophilic and can pass through the plasma membrane. The 11β-HSD1 enzyme catalyzes the conversion of inactive glucocorticoid (cortisone & 11-dehydrocorticosterone) to active glucocorticoid (cortisol & corticosterone), serving as a local amplifier to increase the local concentrations of biologically active glucocorticoid within the cell, whereas 11β-HSD2 catalyzes an opposite reaction. MR activating ligands stimulate MR’s translocation to the nucleus where it can drive downstream transcriptional activity. Figure created with BioRender.com.

In addition to its classical genomic signaling, MR also mediates non-genomic signaling through membrane-associated and cytosolic pathways (53).64 These actions include regulation of epithelial sodium channel (ENaC) activity in renal tubular cells, modulation of voltage-gated calcium channels in vascular smooth muscle, and rapid signaling in cardiomyocytes, skeletal myocytes, and neurons (53). Such responses typically occur within minutes of ligand binding and are largely independent of direct transcriptional regulation (54). These signaling pathways may be particularly important in excitable tissues where ion flux regulates contractility, excitability, and conduction. The non-genomic actions of MR therefore complement its transcriptional functions and may contribute substantially to pathological states associated with mineralocorticoid excess.

Studies demonstrate that chronic MR activation contributes not only to hypertension but also to inflammation, oxidative stress, and tissue remodeling across multiple organ systems (55). Experimental and clinical studies demonstrate that MR dysregulation in renal tubular cells, cardiomyocytes, vascular smooth muscle, and myeloid cells leads to inflammation, oxidative stress, and fibrosis (56, 57). Importantly, these inflammatory effects also influence musculoskeletal tissues. MR signaling in myeloid cells influences cytokine production and osteoclastogenesis and may affect chondrocyte biology (58, 59), suggesting a role for MR dependent immune mechanisms in musculoskeletal physiology and pathology. Together, these findings support the concept that mineralocorticoid excess contributes to multi-organ pathology extending beyond classical cardiovascular dysfunction.

MR signaling in skeletal muscle physiology:

The functions of MR in skeletal muscle are diverse and dynamically modulated by the cellular environment, ligand availability, and crosstalk with other nuclear receptors (60). MR expression has been identified in multiple skeletal muscle cell populations, including myofibers and infiltrating immune cells, with evidence supporting roles in muscle regeneration, inflammation, fibrosis, and repair (60–62). This broad expression pattern suggests that MR signaling may regulate several aspects of skeletal muscle homeostasis beyond classical electrolyte handling. Consistent with this idea, MR signaling has been implicated in muscle regeneration, inflammation, fibrosis, and repair processes (60).

In human skeletal muscle, muscle MR is present as either an intact, full-length form or a spliced variant that lacks exon 5 (63). Differential splicing and post-translational modification serve as tissue-specific mechanisms to fine-tune MR activity (15, 61). The splice variant is also present in tissues including lung, kidney, heart, and hippocampus and has been proposed to exhibit altered ligand binding and transcriptional activity. However, the functional significance of this splice variant remains unclear. (61, 63).

MR antagonism has attracted considerable interest as a therapeutic strategy in DMD because it reduces inflammatory signaling and fibrosis while improving muscle function (60–62). Supporting this idea, Hauck et al. generated a murine conditional knockout (CKO) model of MR in myofibers using the creatine kinase Cre driver (MCK-Cre) which was then crossed with dystrophin-deficient (mdx) mice to generate an MR CKO/mdx model (62). These mice showed functional improvements in respiratory muscle force and muscle fatigue performance as compared to single mutant mdx mice (62). Similarly, Howard et al. treated mdx mice with the MR antagonist spironolactone and showed that MR inhibition reduced inflammatory cytokine expression in the quadriceps muscle and reduced fibrosis in the diaphragm (64). Pharmacological MR antagonism in mdx mice also reduced muscle damage and fibrosis in association with decreased expression of ankyrin repeat domain 1 (Ankrd1), a stress-responsive gene implicated in muscle injury responses (61).

However, the role of MR signaling in skeletal muscle appears highly context dependent. Hauck et al. showed that MR antagonism and knockout in non-dystrophic mouse models impaired regeneration after acute muscle injury in tibialis anterior muscles, with increased expression of cytochrome P-450 group 11, subgroup B, gene 2 (CYP11B2), also known as aldosterone synthase, found in infiltrating CD11b+ myeloid immune cells in the injured tissue (60). Interestingly, myeloid immune cells in DMD also exhibited local aldosterone production by expressing CYP11B2 which is minimal or absent in healthy skeletal muscle (65, 66). This increase of local aldosterone production during muscle injury and dystrophy may contribute to potential MR-mediated immune/fibrotic signaling.

In addition to its role in muscle regeneration, MR signaling has also been linked to metabolic regulation in skeletal muscle. Pharmacological MR inhibition with spironolactone improved glucose tolerance in high-fat diet–fed mice, however, skeletal muscle MR expression was reduced in obesity and did not appear to directly mediate insulin signaling or glucose uptake (67). Given the multifaceted role of MR, further studies in skeletal muscle models are needed to define the role of MR in various muscle cell populations. Clarifying the relative contributions of MR versus GR signaling will be particularly important, as many experimental and therapeutic ligands influence both receptors. Improved understanding of these pathways may help guide strategies aimed at optimizing muscle regeneration, limiting fibrosis, and preserving muscle function in chronic disease states.

Adipose tissue depots and MR:

Obesity is associated with metabolic syndrome and cardiovascular risks, including hypertension, insulin resistance, and dyslipidemia (68, 69). Adipose tissue is now recognized not merely as an energy storage depot, but as an active endocrine and inflammatory organ that contributes to these metabolic disturbances (20, 69, 70). Increased MR expression has been reported in adipose tissue from obese humans and mice (71), and enhanced MR signaling has been linked to adipocyte insulin resistance via disruption of insulin receptor substrate (IRS-1) signaling (72). While mature adipocyte MR depletion was unable to negate high fat diet-induced changes in body composition or glucose homeostasis (73), targeted MR overexpression in adipocytes promoted increased visceral fat accumulation, adipocyte hypertrophy, higher fasting insulin concentrations, elevated levels of triglycerides and cholesterol, and reduced insulin sensitivity (74).

Selective MR activation by aldosterone promoted intracellular accumulation of lipids and enhanced glyceraldehyde 3-phosphate dehydrogenase (G3PDH) activity, driving adipogenic differentiation of 3T3-L1 and 3T3-F442A cells (20, 70). Among adipogenic cell models, 3T3-L1 cells are the most extensively studied and are commonly differentiated into mature adipocytes using glucocorticoids such as dexamethasone (75, 76). Previous studies have suggested that MR signaling contributes to dexamethasone induced adipogenesis despite dexamethasone exhibiting substantially greater affinity for GR than MR (17). Caprio et al. demonstrated that MR knockdown in 3T3-L1 preadipocytes markedly reduced dexamethasone induced adipogenic differentiation as evidenced by decreased G3PDH activity and triglyceride accumulation, whereas GR knockdown had little effect. They attributed this finding to the early and predominant expression of MR during the differentiation process, suggesting that MR mediates the pro-adipogenic actions of dexamethasone under these conditions, despite its lower affinity for the ligand (70). Together, these findings suggest that receptor abundance and timing of expression may influence the relative contribution of MR versus GR signaling during adipogenesis. Furthermore, GR:MR heterodimerization influences downstream gene expression patterns, in addition, transcriptional responses are likely shaped by receptor heterodimerization and interactions with transcriptional coregulators (10, 13, 14, 77).

MR’s role in adipose tissue expansion is further supported by studies demonstrating that increased body mass index (BMI) in humans was associated with increased expression of MR and HSD11B1 genes in visceral fat (20, 78). As mentioned previously, HSD11B1 encodes for 11β-HSD1, the enzyme that catalyzes the conversion of inactive to active glucocorticoids (Figure 2) (79), increasing the intracellular concentration of active glucocorticoids (32). Marked expansion of adipose tissue is a feature of obesity alongside increased pro-inflammatory cytokine release, and research points to MR playing a role in this pro-inflammatory obese state (80). In-vitro studies in which adipocyte MR was selectively activated with aldosterone demonstrated increased production of pro-inflammatory adipokines like IL-6, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-a), and Toll-like receptor 4 (TLR4) (81, 82). These inflammatory pathways subsequently activate nuclear factor κB (NF-κB) and c-Jun N-terminal kinase (JNK), promoting serine phosphorylation of IRS-1 and impaired insulin signaling (82, 83). Interestingly, aldosterone-mediated MR activation also increased leptin production in adipocytes, whereas leptin itself promoted aldosterone biosynthesis and secretion (84). This reciprocal effects suggest the existence of a feed-forward signaling loop linking adipose tissue expansion, aldosterone production, and MR activation during obesity. Moreover, MR activation by glucocorticoids increased reactive oxygen species (ROS) in 3T3-L1 preadipocytes whereas MR blockade improved insulin sensitivity through a reduction in ROS (20, 78, 80). MR activation also contributed to adipokine dysregulation, mitochondrial dysfunction, and oxidative stress, including decreased adiponectin expression, increased inflammatory mediator expression, and enhanced mitochondrial ROS generation; many of these effects were reversed by MR antagonism or MR-targeting siRNA approaches (78, 85).

Like other aldosterone-responsive tissues, 11β-HSD2 is also expressed in white adipose tissue although its expression was reported to be higher in the stromal vascular fraction (SVF) rather than in mature adipocytes (86, 87). Podraza et al. showed lower 11β-HSD2 expression in visceral and subcutaneous fat from obese individuals as compared to controls (88), findings that were subsequently supported by Methlie et al. in subcutaneous adipose tissue samples (86). Consistent with the tissue-specific MR regulation, reduced adipose 11β-HSD2 expression together with increased 11β-HSD1 favor glucocorticoid-driven MR activation during obesity (86). This effect is amplified by the increased 11β-HSD1 found in adipose tissue of obese humans and animals, a phenomenon described as Cushing's disease of the omentum, whereby enhanced local glucocorticoid activation by 11β-HSD1 surpasses 11β-HSD2 activity, promoting sustained MR activation in obesity (89).

Taken together, these studies suggest that MR activation in adipocytes promotes lipid uptake, adipose tissue expansion, adipocyte differentiation, and adipokine release, potentially via MR activation by glucocorticoids. This, then, underlies why MR antagonists like eplerenone and finerenone are being explored for their potential to improve metabolic parameters in obesity and type 2 diabetes (90).

Role of MR in bone health and turnover:

Calcium balance is maintained by intestinal absorption, renal reabsorption, and bone resorption under the regulation of parathyroid hormone (PTH) and vitamin D (91). In the distal convoluted tubule (DCT), calcium balance depends on the electrochemical gradient generated by sodium transport (92). Chronic high MR activation, as in hyperaldosteronism, leads to excessive sodium reabsorption, disrupting the electrochemical gradient needed for calcium reabsorption (93). This leads to calcium loss in the urine (also known as calciuria) and development of renal stones (93, 94). The loss of calcium promotes compensatory secondary hyperparathyroidism to maintain normal calcium levels, which draws calcium from bone (95). Collectively, these systemic effects have been proposed to contribute to the “calcium paradox of aldosteronism,” a phenomenon characterized by concurrent bone loss and nephrolithiasis (95).

The role of the MR in skeletal metabolism is not currently well-defined, but evidence suggests that the MR is well-positioned to affect bone (96, 97). Skeletal research has largely focused on the role that the GR plays in bone (32, 97, 98), in part because of the well-known effects of exogenous glucocorticoid in bone. Glucocorticoid-induced osteoporosis (GIO) is a common clinical issue, affecting at least 50% of people who are on long term glucocorticoid therapy (99). This disorder is the most common type of secondary osteoporosis and the third most common cause of osteoporosis overall (32, 99). Previous studies have demonstrated that fracture risk is directly proportional to the dose and duration of exogenous glucocorticoid treatment (100). An appreciable amount of skeletal research has focused on the negative effect of exogenous glucocorticoid with a broad agreement that these drugs are risk factors for osteoporosis, yet the role of endogenous glucocorticoid in the pathophysiology of osteoporosis is not as well studied and presents with conflicting evidence relating to the roles of GR and 11β-HSD1 / 11β-HSD2 in bone cell populations (43, 97, 98, 101, 102). Notably, 11β-HSD1 expression increases with age, but clinical trials of 11β-HSD1 inhibitors aimed at slowing age-related musculoskeletal decline (e.g., muscle and bone loss), have shown mixed results, highlighting the complexity of endogenous glucocorticoid regulation in skeletal health (103, 104).

Several studies have suggested that endogenous GR-mediated signaling is not the primary driver of bone loss with age, despite the fact that glucocorticoid levels increase with age, and instead suggest that this signaling pathway is vital for bone homeostasis (97, 101, 105). As one example, Rauch et al. first showed that mice with targeted deletion of GR in early osteoprogenitor cells (via conditional GR deletion with Runx2-Cre), had a significant reduction, rather than a gain, in vertebral trabecular bone mass compared to the wildtype control GR-floxed animals, even though no detectable differences in osteoblast and osteoclast numbers were observed (101). Similarly, we developed a model of GR deficiency in Osterix-expressing osteoprogenitor cells (GR-CKOosx), showing that this genetic modification drove (rather than rescued) loss of cortical and trabecular bone mass and increased lipid storage by osteoblasts in a manner that was exacerbated with age (97). Interestingly, however, while GR antagonists such as RU-486 exacerbated this osteoblastic phenotype further, MR antagonists (MRA) including spironolactone and eplerenone reversed lipid storage in GR-CKOosx BMSC derived osteoblasts (97). These findings raise the possibility that MR signaling may contribute to deleterious skeletal phenotypes when GR signaling is impaired, particularly during aging.

Evidence supporting a direct role for MR in bone is quite scarce. Fumoto et al. created CKO models of MR using osteoblast-targeting (Osx) and osteocyte-targeting (DMP-1)-Cre drivers but found little effect of these genetic modifications in young (12-week-old) animals (96). However, pharmacological MRA (eplerenone) treatment increased trabecular bone mass in the tibia of younger mice, and osteocyte-targeted deletion of MR provided partial protection against the harmful skeletal effects of the glucocorticoid prednisolone (96). Because prednisolone exhibits relatively low affinity for MR, these effects may reflect GR–MR crosstalk within osteocytes rather than direct MR activation by prednisolone itself (96). Similarly, MRA treatment (with spironolactone) significantly reduced bone resorption and bone formation markers in postmenopausal women with bilateral primary aldosteronism (106). Bone derived osteocalcin was shown to stimulate adrenal steroidogenesis, although direct interactions between osteocalcin signaling and MR activation remain poorly defined (107). However, many of these studies have not been mechanistically explored, which leaves a knowledge gap regarding the role of MR in musculoskeletal health.

11β-HSD1 expression predominates in skeletal tissues, whereas evidence for endogenous 11β-HSD2 expression in postnatal skeletal cell populations remains limited (19, 43, 102). This concept may be particularly relevant during aging or chronic stress conditions associated with increased glucocorticoid exposure and altered local glucocorticoid metabolism.

BMAT is a specialized fat depot that has been shown to play important roles in bone homeostasis and hematopoiesis (108, 109). The field’s understanding of BMAT’s role in the body is rapidly evolving. BMAT abundance is increased in several disorders associated with skeletal fragility, including osteoporosis, type 1 diabetes, Cushing's disease, estrogen deficiency, anorexia nervosa, and caloric restriction (97, 98, 108). However, BMAT expansion does not always coincide with alterations in bone mass (110–113), and in fact, newer studies show that BMAT has a metabolically supportive role in the bone marrow niche (114). For example, human BMAT has been shown to increase hematopoietic stem cell differentiation and survival (115), and Gunaratnam et al. suggested that BMAT protects osteoblasts from lipotoxicity by its enhanced lipid uptake and storage (116). Similarly, Cawthorn et al. showed that BMAT acted as an endocrine organ during stressful events like caloric restriction, serving as a key source of increased circulating adiponectin (117), and Li et al. recently noted that BMAT acts as a skeletal energy reservoir, promoting lipolysis to support the energetic needs of bone during caloric restriction (118).

Very little is currently known about the role of MR in BMAT, but several studies have investigated the role of GR in this fat depot. We previously reported that BMAT abundance was increased by GR-deficiency in Osx-expressing cells (97, 98). Osterix is expressed in both osteoblasts and bone marrow adipocytes (BMAd), but not white fat adipocytes (118, 119). Recently, a BMAd Cre driver model has been developed (118), allowing more direct investigations into the effects of glucocorticoid in BMAT and bone. Schill et al. used this newly developed BMAd-Cre to conditionally delete the GR in BMAT, finding that this genetic modification did not prevent the expansion of BMAT during caloric restriction (120), which is consistent with our earlier studies showing that targeted deletion of GR in Osx-expressing cells (which includes BMAds) also provided no protection from BMAT expansion during caloric restriction (98). A recent study also found that global knockout of the glucocorticoid-activating enzyme 11β-HSD1 had a sexually dimorphic effect on BMAT expansion during caloric restriction, blunting BMAT expansion in male but not female mice; however decreased GR signaling was not seen in this model, suggesting that circulating levels of active glucocorticoid were still able to impact tissues despite the absence of 11β-HSD1 expression (79). Whether MR contributes to the skeletal or metabolic effects of endogenous glucocorticoid signaling within BMAT remains unknown and represents an important area for future investigation. As discussed previously, MR signaling promotes adipogenic differentiation, and because osteoblasts and bone marrow adipocytes arise from common mesenchymal progenitors, the pro-adipogenic effects of MR activation raise the possibility that MR signaling may influence differentiation within the bone marrow microenvironment. However, direct mechanistic studies in skeletal mesenchymal progenitors remain limited.

Peripheral adipose tissue possesses a local RAAS (121). Because BMAT shares several endocrine and metabolic features with peripheral adipose depots and responds to glucocorticoid signaling (122), local RAAS activity within BMAT is biologically plausible. However, whether BMAT possesses a functionally distinct local RAAS comparable to other adipose depots remains unclear and warrants further investigation.

MR and ectopic calcification:

Ectopic calcification is defined as an abnormal deposition of mineralized matrix composed of calcium phosphate salts like hydroxyapatite in extraosseous tissues (123, 124). Ectopic calcification encompasses a broad spectrum of conditions, ranging from common vascular calcification to rare hereditary calcification disorders (123, 125). Vascular calcification is a pathological process that increases vascular stiffness and reduces compliance, thereby promoting cardiovascular events such as myocardial ischemia and plaque rupture (126). During calcification, vascular smooth muscle cells (VSMC) undergo a trans-differentiation process into osteoblast- and/or chondrocyte-like cells which then produce and deposit extracellular matrix such as collagen type I and hydroxyapatite into the arterial wall (127). The transdifferentiation process producing calcified matrix in VSMCs is mediated by factors including Wnt signaling, the transcription factor Runx2, hypercalcemia, and hyperphosphatemia which promote changes in the local cell environment and increased cell stress (123, 124). These changes have been studied in-vitro where VSMCs were treated with the glucocorticoid dexamethasone to produce vascular calcification. In one study, the VSMCs were treated with GR and MR antagonists to determine which of these two receptors mediated dexamethasone’s harmful effects on ectopic calcification. Interestingly, vascular calcification was ameliorated by the MR antagonist eplerenone whereas the GR antagonist mifepristone failed to reproduce this effect (51, 128), suggesting that MR signaling contributes to the process of vascular calcification.

Aldosterone has also been shown to induce an osteogenic phenotype and promote calcification in VSMCs (39), increasing connective tissue growth factor and collagen deposition by VSMC through galectin-3, neutrophil gelatinase-associated lipocalin (NGAL), endothelin-1-mediated mechanisms, vascular endothelial growth factor receptor 1 (VEGFR1) activation, and NADPH oxidase 1 (Nox1)-mediated pathways (39). In addition, aldosterone activated alkaline phosphatase and reduced autophagy in VSMC, both of which have been reported to act as osteo-inductive signals (39). In these in-vitro and ex-vivo studies, levels of aldosterone vary from 10nM to 100nM representing a range from physiological to supraphysiological levels (129, 130). Studies utilizing supraphysiological levels of aldosterone may have relevance to disease states such as hypertension and hyperaldosteronism but are limited in terms of their relevance to homeostatic physiology (129–131). Although MR-mediated signaling has been shown to induce osteoblast-like transdifferentiation in vascular smooth muscle cells which facilitate ectopic calcification (129), it has not yet been shown whether a similar mechanism could occur in skeletal osteoblasts to affect bone extracellular matrix formation.

BMD and fracture risk associated with MRA use:

The effects of hyperaldosteronism, or excessive aldosterone release from the adrenal glands, can be mimicked in rats through chronic infusion of mineralocorticoids and salt (132). This treatment regimen decreased bone mineral density (BMD) in a manner that was reversed by the MRA spironolactone (133–135). However, despite several studies investigating the effects of MRAs on mice and the availability of different classes of MRAs (136, 137) (Table 1), few studies have directly examined the effects of MRA on bone mineral density or bone metabolism in animal models, particularly in the absence of disease states.

Table 1:

List of pharmacological MR antagonists

MRA	Generation	Nuclear Receptor Specificity	References
Eplerenone	2nd generation	MR antagonist	(Sica, 2015, Yang and Young, 2016)
Spironolactone	1st generation	PR agonist and AR, MR antagonist	(Makhijani et al., 2018, Yang and Young, 2016)
Finerenone	3rd generation	MR antagonist	(Kolkhof et al., 2014)
Drospirenone	3rd generation	MR antagonist	(Caprio et al., 2011)
Dihydropyridines	1st generation	Calcium channel blockers and MR antagonists	(Dietz et al., 2008)
Esaxerenone	3rd generation	MR antagonist	(Wan et al., 2021)
Apararenone (MT-3995)	3rd generation	MR antagonist	(Nakamura and Kawaguchi, 2021)
AZD9977	3rd generation	Partial MR antagonist	(Bamberg et al., 2018)
KBP-5074	3rd generation	MR antagonist	(Pitt et al., 2021)
LY2623091	3rd generation	MR antagonist	(Wang et al., 2017)

AR, androgen receptor; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; MRA, mineralocorticoid receptor antagonist; PR, progesterone receptor

Existing literature suggests that MRAs, which are classified as potassium-sparing diuretics, increase bone mineral density and reduce fracture risk in humans in certain settings. While spironolactone has not been directly employed to prevent bone loss, its use was inversely correlated with fracture risk in men being treated for heart failure (138). Similarly, spironolactone promoted a significant decrease in bone turnover markers in postmenopausal women treated for hyperaldosteronism and improved BMD in patients with hyperparathyroidism secondary to hyperaldosteronism (106) (36). However, MRAs did not significantly alter bone turnover markers in patients with primary hyperparathyroidism (139).

The effect of aldosterone on parathyroid hormone (PTH), and PTH on aldosterone, is not entirely clear (140). Several studies demonstrated that primary aldosteronism was associated with elevated PTH levels via losses of renal tubular calcium and magnesium (141–144). This mechanism is consistent with the altered renal calcium handling associated with excessive MR activation, which alters the electrochemical gradient needed for calcium and magnesium uptake, resulting in their urinary loss (145). Other studies have additionally suggested the existence of bidirectional crosstalk between aldosterone and PTH signaling pathways (146–148). Although MRAs may preserve BMD and reduce fracture risk in hyperaldosteronism, it remains unclear whether these effects occur through direct skeletal actions or indirectly through improvements in systemic physiology (e.g., reduction in blood pressure) (149). MR antagonism also reduces urinary calcium loss, thereby lowering compensatory PTH secretion and indirectly benefiting bone metabolism (150).

The skeletal effects of spironolactone also depend on concomitant hormonal therapies. For example, spironolactone combined with linestrenol was associated with decreased BMD in young women treated for hyperandrogenemia, whereas combination therapy with ethinylestradiol and desogestrel had no detectable effect on BMD (151, 152). In addition, spironolactone was reported to be as effective as bisphosphonates or estrogen-progestin therapy in preventing gonadotropin-releasing hormone antagonist-induced bone loss (153). Another MRA, drospirenone, blocks both the androgenic receptor (AR) and MR, but lacks any estrogenic, glucocorticoid, or anti-glucocorticoid activity (154). Drospirenone also exhibits high affinity for MR (154). Drospirenone, when combined with oral contraceptives, had a beneficial effect on bone turnover markers in young women (155).

Finerenone is a non-steroidal MR antagonist with a non-lipophilic structure (156–158). Compared with steroidal MRAs, finerenone exhibits reduced renal accumulation and distinct cofactor recruitment properties while retaining potent anti-fibrotic and cardio-renal protective effects (159, 160). Its bulky structure prevents recruitment of transcriptional co-factors, suppressing MR signaling with potency comparable to spironolactone but greater selectivity than eplerenone (156). Although direct clinical evidence regarding finerenone and skeletal outcomes is lacking, its tissue-selective MR antagonism makes it an interesting candidate for future investigation (161, 162). MRAs additionally influence skeletal metabolism indirectly through altered potassium and calcium handling (163–165).

MRAs are used clinically for treating hypertension (160). In addition to exerting an effect on the kidneys, spironolactone and eplerenone both cross the blood-brain barrier and decrease peripheral sympathetic tone which in turn aids in lowering blood pressure (166–169). Excessive activation of the MR, typically driven by elevated aldosterone levels, is well-established as a key factor in mineralocorticoid hypertension and increased sympathetic tone. However, studies also suggest broader systemic effects of MR activation, particularly concerning bone and muscle health (170). Multiple epidemiological studies across diverse populations have reported associations between hypertension and reduced BMD, increased bone loss, or elevated fracture risk. (171–175). Moreover, hypertension results in abnormalities of calcium metabolism, such as the increased excretion of calcium associated with salt intake (176–178). Excessive MR activation may further contribute to these abnormalities in mineral metabolism (179). Historically, this mechanism was considered relatively uncommon prior to broader use of aldosterone-to-renin ratio screening for primary aldosteronism (179). In addition to altered renal calcium handling, heightened sympathetic tone represents another pathway by which mineralocorticoid hypertension impairs skeletal health (135). Increased sympathetic outflow enhances norepinephrine release, activating β2-adrenergic receptors on osteoblasts, this suppresses bone formation, increases RANKL expression, and promotes osteoclastogenesis, leading to net bone loss (180). Supporting this, β-blocker therapy prevents bone loss in animal models and has been associated with improved BMD in some human studies (181). Given that MR activation drives sympathetic nervous system overactivity (182, 183), part of the bone-sparing effects of MRAs occur through suppression of sympathetic signaling rather than direct skeletal MR inhibition.

Thus, it is possible that the beneficial effects of MRAs on bone in hypertensive models could be secondary to normalization of blood pressure or increases in serum calcium levels. However, conflicting evidence remains regarding the relationship between hypertension and skeletal health, as several studies have reported no significant association between blood pressure and bone mass (184–186). Therefore, further mechanistic studies on the direct effects of MRAs on bone in normotensive and hypertensive models are needed to better understand this relationship.

Crosstalk between the adrenal gland and other organs relevant to calcium homeostasis

Organ crosstalk occurs when two or more organ systems within the body communicate in a unidirectional or reciprocal manner (187). This process is mediated by signaling factors like hormones, cytokines, and growth factors (187), and often facilitates homeostasis; however, aberrant regulation in one organ could adversely affect another. The adrenal gland influences bone (and other organs) through the secretion of hormones; however, the resultant action depends on the timing and amount of hormone secretion, expression level of hormone-metabolizing enzymes in the target tissue, and the affinity and expression level of the hormone’s target receptors (30). As mentioned previously, the adrenal gland’s outer cortex produces aldosterone in the zona glomerulosa, glucocorticoid steroid hormones like cortisol in the zona fasciculata, and androgens in the zona reticularis, while its inner medulla produces catecholamines such as epinephrine (30). Excessive MR activation has well-established effects on the cardiovascular system, particularly in promoting hypertension (39, 51, 188, 189). Multiple studies have reported associations among hypertension, altered renal calcium handling, hypercalciuria, and secondary hyperparathyroidism, suggesting a potential link between MR signaling and systemic calcium regulation (144, 172, 176, 177, 179, 190, 191). While serum calcium is tightly maintained within a narrow physiological range, even at the expense of skeletal stores, MR activation influences this balance indirectly (49). One proposed mechanism involves MR-dependent alterations in renal calcium handling (140). MR additionally regulates calcium and ion channel activity in neurons, cardiomyocytes, and vascular smooth muscle cells; however, these effects primarily influence cellular excitability and contractility rather than systemic calcium balance (29, 49). Collectively, these findings suggest that dysregulated MR signaling in cardiovascular disease influences calcium homeostasis in ways that extend beyond the cardiovascular system itself (192, 193). Excess glucocorticoid exposure impairs intestinal calcium absorption, inhibits osteoblast differentiation, promotes osteoblast and osteocyte apoptosis, and increases bone resorption, ultimately reducing bone mineral density (194–196).

Calcium acts as a ubiquitous intracellular signaling molecule, and disturbances in calcium homeostasis therefore affects multiple tissues and organ systems (197). However, important mechanistic gaps remain, particularly because many studies investigating MR signaling and calcium regulation have relied heavily on in vitro models. Additional in vivo studies are therefore needed to better define the contribution of MR signaling to systemic calcium homeostasis.

Glucocorticoid-induced osteoporosis is sometimes associated with a secondary rise in the level of PTH secreted from the parathyroid gland (198), and several studies relate this observation to a compensatory response of the body to glucocorticoid induced reduction in serum calcium levels (192, 198). However, accurate characterization of hormone secretion patterns is challenging, requiring intensive blood sampling for accurate results (192). Using such methodology, Bonadonna et al. demonstrated that chronic glucocorticoid treatment altered PTH secretory dynamics in humans, decreasing tonic secretion while increasing pulsatile PTH release (192). This analytical approach estimates parameters including tonic secretion rate, pulsatile secretion frequency, and hormone half-life from serial plasma measurements. Tonic PTH secretion was reduced in glucocorticoid-treated individuals compared with controls, whereas the proportion of PTH released in a pulsatile manner was substantially increased (192). Since pulsatile secretion of PTH is highly sensitive to changes in ionized calcium and glucocorticoids reduce calcium absorption and increase urinary excretion, this promotes an indirect mechanism that triggers PTH secretion from the parathyroid glands in a pulsatile manner (192, 199–201). Schilling et al explored the effects of dexamethasone on PTH secretion and found differing effects between in-vitro and in-vivo models (202). Dexamethasone induced dose-dependent inhibition of PTH secretion in the WCS 256 rat mammary carcinoma cell line; however, the cells lost this ability when transplanted into female Wistar rats that received treatment with dexamethasone suggesting an indirect systemic effect (202). These observations further highlight the complexity of endocrine crosstalk between glucocorticoid signaling, calcium regulation, and parathyroid hormone secretion. Such mechanisms also contribute to the therapeutic efficacy of intermittent PTH treatment in glucocorticoid-induced osteoporosis (203).

Conclusions

The physiological and pathological roles of MR outside of its classical renal functions are increasingly recognized, although much work remains to define the role of MR in bone. Over the past several decades, studies have demonstrated that excessive MR activation contributes to cardiovascular disease, obesity associated metabolic dysfunction, glucocorticoid-induced skin atrophy, skeletal muscle degeneration, and fibrosis (60, 61, 64, 204–206). However, the role of MR in skeletal diseases remains understudied, particularly relative to GR signaling (Figure 3).

Figure 3: Downstream effects of MR signaling in skeletal muscle, adipose tissue, and skeletal tissues.

Adrenal glands secrete glucocorticoid which are capable of activating MR in peripheral tissues including skeletal muscle, adipose tissue, bone marrow adipose tissue (BMAT) and bone. While the effects of MR activation have been reported in several studies for skeletal muscle and adipose tissue, the effects of glucocorticoid-MR signaling on bone and BMAT physiology remains unclear at present. Figure created with BioRender.com.

Glucocorticoid signaling through GR has been suggested to be important for bone health as skeletal integrity decreased with GR deletion (97, 98). However, these findings also raise the possibility that unopposed MR signaling may contribute to skeletal deterioration when GR signaling is impaired. Several studies suggest that pharmacological MRAs increased trabecular bone mass in rodents, but whether MR activation mediates age-related bone loss remains unclear. MR inhibition was associated with reduced fracture risk in aged men, although the direct impact of the drug on bone (as compared to an overall improvement in health status) was not addressed in this study (96, 138).

Because MR signaling influences calcium and magnesium homeostasis, it may affect skeletal physiology either directly through bone cells or indirectly through regulation of PTH secretion and systemic mineral balance. Mechanistic studies that directly examine MR action in different bone cell populations are needed to determine how MR interacts with GR, PTH, local glucocorticoid metabolism, and BMAT to understand the full effect of MR-mediated signaling in the skeleton. Further studies, using currently available genetic tools and drugs as well as novel models, are needed to clarify the role of MR in musculoskeletal health in part by identifying specific mechanisms of glucocorticoid-MR signaling and transcriptomic targets that are regulated by GR, MR, or both. Taken together, the findings presented here suggest new avenues to investigate factors controlling bone homeostasis; glucocorticoid-mediated MR signaling, and the bone’s role as an endocrine organ. Improved understanding of these pathways may ultimately identify novel therapeutic targets for osteoporosis and other musculoskeletal diseases.