Endocrinology Vol. 148, No. 4 1498-1501
Copyright © 2007 by The Endocrine Society
Mineralocorticoid Receptors and Vascular Inflammation: New Answers, New Questions
Patrick M. Blanner,
Ruteja A. Barve and
Charles W. Bolten
Pfizer Global Research & Development, St. Louis Laboratories, Chesterfield, Missouri 63017
Address all correspondence and requests for reprints to: Charles W. Bolten, Pfizer Global Research & Development, 700 Chesterfield Parkway, Chesterfield, Missouri 63017. E-mail: charles.w.bolten{at}pfizer.com.
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Introduction
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Reports on the deleterious effects of mineralocorticoids plus salt on the vasculature began in the 1940s but drew little attention until the 1990s (1), a decade that closed with the 1999 publication of the full results from the Randomized Aldactone Evaluation Study (RALES) (2). Pitt et al. (2) reported that patients with severe heart failure taking spironolactone (Aldactone), a steroidal mineralocorticoid receptor (MR) antagonist derived from progesterone, showed a 30% improvement in all-cause mortality over standard of care. The benefit of MR blockade was reinforced when the selective MR antagonist eplerenone (Inspra) was shown to be cardioprotective in patients with acute myocardial infarction heart failure (3). The data were now clear; activated MR could inflict deleterious effects on the cardiovascular system and intervention with an MR antagonist saved lives. Seven years after the publication of RALES the fundamental question remains: how? The gene expression experiments published by Géza Fejes-Tóth and Anikó Náray-Fejes-Tóth in this issue of Endocrinology (4) provide the most compelling information to date as to the first events that lead to the cascade of inflammation and cardiovascular remodeling that is associated with inappropriate MR activation and point to interesting possibilities that these mechanisms may have broader implication in human diseases.
In reflecting on the slow progress of research into the mechanisms through which MR exerts damaging effects on the heart, some discussion of the technical issues that have made study of this receptor and interpretation of some published data relatively difficult is warranted. We and others have observed that the mRNA for MR is measurable in many mammalian cells in culture including those derived from macrophage, cardiomyocyte, epithelial, endothelial, and vascular smooth muscle cells but that the protein is far more selectively expressed. Thus, demonstration of receptor mRNA in vitro where MR gene regulation is reported is not convincing. Documenting MR expression is further problematic because commercially available antibodies have been of variable quality. In addition to issues related to expression, aldosterone’s ability to activate both the mineralocorticoid and glucocorticoid receptors (GR) (5) has been underappreciated in the literature. As noted by Fejes-Tóth and Náray-Fejes-Tóth, many authors have employed high concentrations of aldosterone to demonstrate gene expression changes. Although this does not exclude the possibility that any findings could, at least in part, be MR mediated, it does mean that GR activation by aldosterone would need to be excluded. Collectively, these issues raise important questions that should be asked before examining the potential MR-regulated transcripts identified in any paper:
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Is MR expressed in the system being studied?
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Fejes-Tóth and Náray-Fejes-Tóth’s overexpression of MR in H9C2 cells can be accepted confidently because of their use of a monoclonal antibody from the highly regarded Gomez-Sanchez laboratory (6), whose investigators recently published an MR tissue distribution based on immunohistochemistry. Beyond showing that MR protein is expressed, Fejes-Tóth and Náray-Fejes-Tóth demonstrate 3H-aldosteone binding in MR-expressing cells that is competed with cold aldosterone but not excess RU486, a steroidal antagonist of both the progesterone and glucocorticoid receptors, indicating that the specific binding observed is to MR.
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Are concentrations of aldosterone appropriate for MR vs. GR activation used?
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At higher concentrations aldosterone is perfectly competent to bind as an agonist ligand for the promiscuously expressed GR (5). Fejes-Tóth and Náray-Fejes-Tóth report microarray data for cells treated with 1 nM aldosterone, an appropriate maximal dose for aldosterone activation of MR, and extend their array findings to dose response for a representative gene where sub-nanomolar activity is observed. These concentrations are consistent with MR- vs. GR-mediated activity.
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Are established MR target genes regulated by aldosterone in the experimental model?
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Fejes-Tóth and Náray-Fejes-Tóth show an increase in the prototype MR target gene serum and glucocorticoid-regulated kinase (7) with 1 nM aldosterone, an effect that is not observed in clones not expressing MR. Additionally, microarray analyses revealed that glucocorticoid-induced leucine zipper protein, a more recently documented MR target gene (7), is also regulated by aldosterone in this system. Although it remains possible that the overlap between MR-regulated genes in nonepithelial vs. epithelial cells could be small and that known target genes linked primarily to sodium transport may not be regulated in cardiomyocytes, demonstration that some established target genes are regulated as expected increases confidence that the system is working appropriately.
Having established that the gene expression studies conducted by Fejes-Tóth and Náray-Fejes-Tóth are of high technical quality and that most effects reported are likely mediated by MR, a discussion of the potential implications of their findings is warranted. Using an MR-expressing cardiomyocyte in vitro model, the authors have identified putative aldosterone-regulated genes with potential roles in the cardiovascular pathophysiology associated with aldosterone. Included in their discussion are genes involved in extracellular matrix regulation [a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), hyaluronic acid synthase-2, tenascin-X, plasminogen activator inhibitor-1 (PAI-1), and urokinase-type plasminogen activator receptor], vascular tone (regulator of G protein signaling-2 and adrenomedullin), and inflammation (orosomucoid-1).
Of the genes identified by Fejes-Tóth and Náray-Fejes-Tóth (4), orosomucoid-1 (Orm-1 or 
-acid glycoprotein) demonstrated the highest level of induction upon aldosterone administration (100-fold higher after 2-h and 1000-fold after 24-h treatment with 1 nM aldosterone). Orm-1 is an acute-phase plasma protein with detected expression in a number of tissues including liver, kidney, heart, brain, and lung (8). Orm-1 is induced by glucocorticoids; the proinflammatory cytokines IL-1ß, IL-6, and TNF; as well as other inflammatory mediators of the acute-phase response (9, 10). Numerous studies have established an inflammatory component in cardiovascular disease, type 2 diabetes, as well as increased PAI-1 expression and vascular reactivity (11). Orm-1 has been demonstrated to bind PAI-1, resulting in the stabilization of PAI-1 conformation and thus preserving its inhibitory activity (12). Elevated plasma levels of Orm-1 are associated with cardiovascular disease and type 2 diabetes and are therefore regarded as cardiovascular risk factors (13). This involvement in multiple inflammatory-mediated cardiovascular responses strongly implicates Orm-1 in the inflammatory effects of aldosterone.
Tenascin-X (TN-X) is a member of the "matricellular" family of extracellular matrix proteins, of which tenascin-C, osteonectin, osteopontin, thrombospondin-1, and thrombospondin-2 are also members (14, 15). Matricellular proteins have significant impact on cell function and cell-matrix interactions while having no direct structural role within the extracellular matrix (14). A major site of TN-X expression is the heart, and this expression is up-regulated during tissue injury and remodeling (15, 16). Further studies suggest that TN-X is involved in collagen matrix assembly, as evidenced by the fact that TN-X deficiency in humans (Ehlers-Danlos syndrome) and knockout mice exhibit decreased collagen content in the skin (14). These findings suggest that the aldosterone-induced up-regulation of TN-X expression in cardiomyocytes plays a role in the adverse effects (extracellular matrix remodeling and cardiac fibrosis) of aldosterone in the heart.
Another extracellular matrix-related gene the authors have identified to be up-regulated in cardiomyocytes is ADAMTS1. A member of the ADAMTS family of extracellular proteases, ADAMTS1 is involved in inflammation, angiogenesis, and development of the urogenital system (17). Further studies have demonstrated that ADAMTS1 plays a role in extracellular matrix remodeling through its cleavage of tissue factor pathway inhibitor-2, resulting in altered distribution of this protein within the extracellular matrix (17). A role for ADAMTS1 in atherosclerosis has been established by the finding of increased ADAMTS1 expression in atherosclerotic lesions, as well as migrating vascular smooth muscle cells (18). ADAMTS1 has also been implicated in myocardial infarction, as increased expression of ADAMTS1 was demonstrated in both the endothelium and cardiomyocytes immediately surrounding the infarct zone (19). Together, these findings suggest a role for ADAMTS1 in both the vascular inflammatory and fibrotic effects of aldosterone in the cardiovascular system.
It is logical to examine genes identified in the experiments by Fejes-Tóth and Náray-Fejes-Tóth (4) to look for insight into diseases where we know MR plays an important role. It may also be fruitful to take the opposite approach and consider other diseases that are associated with these putative MR target genes and ask whether MR might be implicated. To our knowledge, little is known about MR expression in chondrocytes or other cells of the joint. The receptor has been reported in some fibroblasts and macrophage, but no papers demonstrate expression in cells of these types in the synovium. However, the result of Fejes-Tóth and Náray-Fejes-Tóth showing acute aldosterone down-regulation of hyaluronan synthase-2 hints at an interesting link to osteoarthritis, a debilitating disease of inflammation, extracellular matrix remodeling, and cartilage degradation. Hyaluronan synthase-2 expressed in synovial fibroblasts produces high-molecular-weight hyaluronan, a major molecule in joint fluid that plays a crucial role in the maintenance of joint homeostasis. Interestingly, the concentration and average molecular weight of hyaluronan are reduced in osteoarthritic and other diseased joints compared with normal individuals (20, 21, 22), making it interesting to wonder whether HAS2 is a direct, negatively regulated MR target gene, as the data of Fejes-Tóth and Náray-Fejes-Tóth imply, this could be MR mediated. Interestingly, there is precedence for negative regulation of direct target genes in osteoporosis by the closely related glucocorticoid receptor (23).
In addition to an acute decrease in HAS2 expression, Fejes-Tóth and Náray-Fejes-Tóth report aldosterone-mediated induction of ADAMTS1 within 30 min, an effect that increases through their 24-h experiment. Although a connection between ADAMTS1 and human osteoarthritis has not been made, this enzyme has been shown to degrade aggrecan (24), the major proteoglycan of cartilage and an important component of the extracellular matrix surrounding cartilage chondrocytes (25). Because aggrecan depletion leading to detrimental changes in the mechanical properties of cartilage is an important step in the progression of disease (26), other ADAMTS family members known to cleave aggrecan are being pursued as osteoarthritis targets (26, 27). Consequently, further examination of MR-mediated ADAMTS1 expression could prove to be important in osteoarthritis.
The proinflammatory effects of activated MR could also be important in airway diseases. Chronic obstructive pulmonary disease (COPD) is a disease featuring a chronic cycle of injury, inflammation, abnormal wound repair and remodeling, producing long-term changes to the normal tissue architecture and reducing lung function (28). A cursory examination would suggest parallels between COPD and the MR-mediated progression of inflammation and fibrosis seen in cardiac and renal diseases. Beyond the possibility that MR could be involved in lung inflammation, fluid balance, a task traditionally assigned to epithelial MR, is essential to normal airway function and increased Na+ absorption can be detrimental (29). Inappropriate epithelial sodium channel (ENaC) activity can cause dehydration of the airway surface liquid, mucus thickening, and poor mucus clearance, which is postulated to contribute to the progression of diseases such as cystic fibrosis, chronic bronchitis, and COPD (30). Accordingly, the ENaC inhibitor amiloride has shown some utility in increasing airway surface liquid volume and improving mucus clearance (31). In light of the data of Fejes-Tóth and Náray-Fejes-Tóth suggesting that MR directly regulates genes involved in inflammation and fibrosis, it might be logical to examine whether an MR antagonist is efficacious in airway diseases that feature increased Na+ transport, inflammation, and extracellular matrix remodeling, where it might have the ability to impact all of these processes (Fig. 1
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FIG. 1. Model for MR involvement in diseases of inflammation and fibrosis. At the molecular level, MR activation by aldosterone or cortisol leads to changes in the transcription of the genes identified in red. Contained in the dotted red box are the newly identified putative MR-regulated genes, whereas ENaC, serum and glucocorticoid-regulated kinase 1 (SGK1), glucocorticoid-induced leucine zipper protein (GILZ), and CHIF represent previously confirmed MR target genes. The increased expression of these genes impacts the cellular responses described in the gray box, namely tissue remodeling, fibrosis, inflammation, and fluid balance. Adverse effects of MR-regulated genes on these cellular processes have been demonstrated in cardiovascular disease. Inflammatory and airway disease, depicted in the dotted black boxes, may prove to be additional areas impacted by activation of MR. uPAR, Urokinase-type plasminogen activator receptor; CHIF, corticosteroid hormone-induced factor.
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Realization in the 1990s that MR antagonism protects the cardiovascular system from damaging inflammation and remodeling provided a new focus to those working on aldosterone mechanism of action. It is therefore surprising that there has not been a single direct target gene strongly linked to the nonepithelial pathophysiology associated with MR activation. The well-conceived and -executed study reported by Fejes-Tóth and Náray-Fejes-Tóth (4) in this issue of Endocrinology offers intriguing new candidate target genes that could provide the missing link, and because many of these genes have been implicated in other diseases of inflammation and fibrosis, suggests the possibility that MR could play a broad role in inflammatory disease.
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Acknowledgments
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The authors thank Steve Roberds, Alex Hromockyj, and John Minnerly for assistance with this commentary.
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Footnotes
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Abbreviations: ADAMTS, A disintegrin and metalloproteinase with thrombospondin motifs; COPD, chronic obstructive pulmonary disease; GR, glucocorticoid receptor; ENaC, epithelial sodium channel; MR, mineralocorticoid receptor; Orm-1, orosomucoid-1; PAI-1, plasminogen activator inhibitor-1; TN-X, tenascin-X.
Received January 24, 2007.
Accepted for publication February 6, 2007.
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Introduction
Is MR expressed in...
