The Mineralocorticoid Receptor Is a Constitutive Nuclear Factor in Cardiomyocytes due to Hyperactive Nuclear Localization Signals

Abstract

The mineralocorticoid receptor (MR), a member of the nuclear receptor family, mediates the action of aldosterone in target epithelia, enhancing sodium reabsorption. In addition, MR may have other physiological functions in nonepithelial tissues. Altered expression or inappropriate activation of cardiac MR is directly linked to the development of cardiac fibrosis, and MR blockade is beneficial for the treatment of heart failure. However, the physiological role, activation status, and target genes of MR in the heart are poorly known. Because ligand-free steroid receptors are typically cytoplasmic and translocate to the nucleus upon ligand binding, we examined the subcellular localization of MR under different corticosteroid levels using subcellular fractionation and immunostaining. Our results demonstrate that MR is a chromatin-bound factor in mouse left ventricle and in a cultured model of cardiomyocytes, HL-1 cells, regardless of circulating corticosteroid levels. Immunohistochemical localization of MR in human heart confirms the subcellular localization pattern. Mutation of nuclear localization signals (NLSs) demonstrates that MR constitutive nuclear localization mainly depends on the synergistic contribution of NLS0 and NLS1. Constitutive nuclear localization in HL-1 cells can be reverted by cotransfection of heat shock protein 90. Heat shock protein 90 expression levels in the mouse heart and HL-1 cells are lower than those found in other tissues, suggesting that low levels of cochaperones render MR NLSs hyperactive in cardiomyocytes. Even though MR is constitutively nuclear, corticosteroids still control the transactivation properties of the receptor in a model promoter, although other MR ligand-independent activities cannot be excluded.

Primary Hyperparathyroidism With Concurrent Primary Aldosteronism

“…This finding was confirmed with immunoblotting on nuclear and cytosol protein fractions ( Figure S1D), as well as at the mRNA level in the parathyroid adenoma of the index case ( Figure S1E and S1F). A similar predominantly nuclear localization of the MR has been found recently in other tissues, including the heart, 28,29 that are now regarded as targets of aldosterone action.…”

Phosphorylation of Mineralocorticoid Receptor Ligand Binding Domain Impairs Receptor Activation and Has a Dominant Negative Effect over Non-phosphorylated Receptors

“…In the absence of ligand the vast majority of cells showed a predominantly cytosolic MR localization (Fig. 2, A and C) as previously described (7,8). Mutations S839D or S839E did not change naïve MR subcellular localization (Fig.…”

“…Semiquantitative analysis of subcellular distribution in the absence of aldosterone was performed as previously described (7,8,26). Briefly, cells were fixed, images were taken under a confocal microscope, and at least 75 cells per condition were scored into five categories (N, exclusive nuclear localization; N Ͼ C, predominant nuclear localization; N ϭ C, even distribution throughout cytosol and nucleus; N Ͻ C, predominant cytosolic localization; C, exclusive cytosolic localization).…”

Neutrophil Gelatinase-Associated Lipocalin Is a Novel Mineralocorticoid Target in the Cardiovascular System

“…22 Cell treatments are indicated in legends of Figure 4. Cell transfection and chromatin immunoprecipitation are described in the supplementary Methods section.…”

“…To evaluate the mechanism of NGAL regulation by MR activation, we used the mouse cardiomyocyte cell line HL-1 cells that do not express detectable levels of endogenous MR activity 22 and that were transiently transfected with mouse MR. Aldo produced a 6-fold increase in NGAL mRNA levels in HL-1 cells transfected with mouse MR but not in HL-1 cells transfected with empty vector (Figure 4A). Cotransfection of mouse MR and NGAL-luciferase reporter construct (using the Ϫ794 to ϩ36 NGAL promoter region) that contains a hormone response element ( Figure 4B), followed by Aldo treatment, resulted in a 4-fold increase in luciferase activity ( Figure 4C) that was blocked by the MR antagonist spironolactone ( Figure 4C).…”